Geobacillus stearothermophilus alpha amylase (amys) variants with improved properties

ABSTRACT

Described are variants of a parent α-amylase that exhibits an alteration in at least one of the following properties relative to said parent α-amylase: specific activity, substrate specificity, substrate binding, substrate cleavage, thermal stability, pH-dependent activity, pH-dependent stability, oxidative stability, Ca2+ dependency, pI, and wash performance. The variants are suitable for starch conversion, ethanol production, laundry washing, dish washing, hard surface cleaning, textile desizing, and/or sweetener production.

PRIORITY

Pursuant to 35 U.S.C. §120 and 35 U.S.C. §119(e), the present application claims the benefit of and priority to U.S. patent application Ser. No. 12/477,028, filed Jun. 2, 2009 and to U.S. Provisional Patent Application Ser. No. 61/059,423, filed Jun. 6, 2008, each of which is incorporated by reference.

TECHNICAL FIELD

Described are variants of a parent α-amylase that exhibits an alteration in at least one of the following properties relative to said parent α-amylase: specific activity, substrate specificity, substrate binding, substrate cleavage, thermal stability, pH-dependent activity, pH-dependent stability, oxidative stability, Ca²⁺ dependency, pI, and wash performance. The variants are suitable for starch conversion, ethanol production, laundry washing, dish washing, hard surface cleaning, textile desizing, and/or sweetener production.

BACKGROUND

Alpha (α)-amylases (α-1,4-glucan-4-glucanohydrolases, E.C. 3.2.1.1) constitute a group of enzymes, which catalyze the hydrolysis of starch and other linear and branched 1,4-glucosidic oligo- and polysaccharides. α-amylases can be used commercially in the initial stages of starch processing (liquefaction); in wet corn milling; in alcohol production; as cleaning agents in detergent matrices; in the textile industry for starch desizing; in baking applications; in the beverage industry; in oilfields in drilling processes; in deinking of recycled paper and in animal feed.

Although currently available α-amylases have been used with some success in these applications, there remains a need for α-amylases with increased specific activity, tailored substrate specificity, improved thermal, pH, and oxidative stability, and reduced Ca²⁺ dependency.

SUMMARY

In one aspect, novel α-amylolytic variants (mutants) of a SPEZYME® Xtra or AmyS-like α-amylase, are provided, in particular variants that exhibit altered properties which are advantageous in connection with the industrial processing of starch (starch liquefaction, saccharification, cleaning, and the like).

Such alterations in properties may be achieved by introducing mutations into a parental α-amylase that affect, e.g., specific activity, substrate specificity, substrate binding, the substrate cleavage pattern, thermal stability, the pH/activity profile, the pH/stability profile, stability towards oxidation, Ca²⁺ dependency, and other properties of interest. For instance, the alteration may result in a variant which, as compared to the parent Spezyme Xtra-like α-amylase, has a reduced Ca²⁺ dependency and/or an altered pH/activity profile and/or thermostability.

In some embodiments, the variants are based on the parent Geobacillus stearothermophilus α-amylase, or have a specified degree of amino acid sequence identity to this α-amylase, e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even 99%. In other embodiments, the variants are based on related parent α-amylase, e.g., those which have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even 99% amino acid sequence identity to Geobacillus stearothermophilus α-amylase.

In some embodiments, a variant polypeptide having α-amylase activity and at least one altered characteristic that improves enzyme performance is provided, the variant polypeptide comprising an amino acid sequence having at least 60% amino acid sequence identity to a parental α-amylase polypeptide selected from AmyS (SEQ ID NO: 1) or a truncated variant of AmyS (SEQ ID NO: 2), and having at least one of the following mutations at an amino acid residue corresponding to that of the parental α-amylase polypeptide as determined by aligning the variant polypeptides with the parental polypeptide, wherein the mutation changes the amino acid residue from that of the parental polypeptides:

a) a substitution that introduces a positively charged amino acid residue one or more positions selected from the group consisting of D19, N28, E29, Q86, Q89, Q97, N224, N271, N281, D306, D318, Q319, Q358, D393, Q443, and D458;

b) a substitution that introduces one or more of the amino acid residues selected from the group consisting of 74A, 115L, 124K, 124R, 132A, 132C, 135A, 145A, 146A, 148A, 148N, 159A, 159C, 159D, 159E, 159F, 159G, 159H, 159K, 159L, 159N, 159R, 159S, 159T, 159V, 169A, 169L, 169M, 169Y, 179A, 181A, 181C, 181D, 181E, 181L, 181P, 181Q, 181V, 181Y, 242A, 242D, 242E, 242Q, 261L, 271A, 271V, 278A, 278H, 278K, 278N, 278R, 281A, 281L, 281M, 302D, 302M, 304D, 304E, 304M, 321A, 321H, 321Q, 321R, 333Q, 378D, 378N, 378R, 382D, 398A, 418A, 418M, 418N, 420A, 421R, 432A, 432D, 432L, 432M, 432N, 432Q, 432R, 432Y, 437D, 437G, 437H, 437L, 437M, 437Y, 446A, 446Y, 454A, 464Q, 464Y, 474A, 474E, 474K, 474L, 474M, 474N, 474P, 474Q, 474R, 474S, and 474V;

c) a substitution that introduces one or more of the amino acid residues selected from the group consisting of 6I, 6N, 6Q, 6T, 6V, 14T, 16F, 25A, 25C, 25G, 25Q, 27M, 36Q, 36S, 39G, 39V, 50I, 50L, 50M, 50N, 50Q, 52S, 53T, 67N, 67S, 80D, 80I, 90E, 133P, 133V, 137M, 137S, 141E, 141I, 141L, 141M, 141Q, 141R, 141S, 141V, 150E, 151I, 152G, 155S, 155Y, 168W, 173T, 188P, 193F, 193K, 193L, 193Y, 213L, 213M, 213V, 217Q, 220P, 220Q, 220R, 220S, 220V, 221I, 221S, 249E, 250F, 250I, 250M, 252L, 253Y, 254E, 254F, 254T, 254V, 255F, 255K, 255W, 257L, 257M, 257S, 257V, 258D, 258G, 258H, 258K, 258Q, 258T, 258V, 268F, 274W, 283M, 283N, 283V, 285E, 285Q, 293G, 293K, 294W, 301F, 301I, 301P, 301R, 301T, 301W, 309D, 309V, 312H, 312S, 312V, 312Y, 313G, 313H, 313I, 313L, 313S, 313V, 318T, 338A, 338C, 338G, 338M, 338T, 339K, 339T, 339V, 340A, 340M, 340Q, 340T, 343C, 343I, 343P, 343R, 343Y, 345I, 345Q, 369I, 369T, 370G, 375T, 385T, 386K, 394L, 394V, 400A, 400N, 400V, 402H, 4021, 402T, 402V, 402W, 403A, 403E, 403G, 403Q, 403R, 403T, 403V, 404C, 404E, 404G, 404I, 404V, 419A, 419C, 419M, 419T, 422E, 422G, 433A, 433H, 433I, 433K, 433L, 433M, 433V, 433Y, 442A, 442G, 442N, 442R, 442S, 442T, 442V, 442W, 442Y, 445G, 445I, 445N, 445T, 445V, 445W, 447I, 447N, 447Q, 447W, 447Y, 448C, 448F, 448G, 448H, 448I, 448N, 448Y, 450C, 450H, 450M, 450N, 450R, 450S, 450T, 450W, 455G, 455I, 455P, 455V, 463A, 463M, 463S, 463T, 463V, 463W, 465G, 465I, 465K, 465N, 465T, 465V, 469D, 469W, 469Y, 471I, 471V, 473G, 473Y, 476A, 476G, 476L, 476M, 476N, and 476T

d) a substitution that introduces one or more of the amino acid residues selected from the group consisting of 124N, 125A, 125K, 125N, 130A, 130S, 159A, 159D, 159E, 159G, 159H, 159K, 159L, 159N, 159R, 159S, 159T, 166F, 166G, 166H, 166S, 166Y, 169L, 179A, 179P, 180A, 180D, 180H, 180K, 180L, 180N, 180T, 180V, 180Y, 181A, 181D, 181E, 181G, 181P, 181R, 181S, 181V, 187A, 187C, 187K, 187N, 187P, 187Q, 187R, 187S, 242H, 242N, 278H, 278K, 278N, 278R, 281M, 302D, 304M, 304Y, 321H, 321Q, 321R, 333Q, 432Q, 437Y, 446A, 474Q, and 474S,

e) a substitution that introduces one or more of the amino acid residues selected from the group consisting of 6A, 6D, 6E, 6H, 6I, 6K, 6L, 6M, 6N, 6P, 6Q, 6R, 6S, 6T, 6V, 6W, 6Y, 13K, 14F, 14T, 14Y, 15A, 15D, 15E, 015G, 15H, 15K, 15N, 15P, 15Q, 15R, 15S, 15T, 15W, 16A, 16E, 16G, 16H, 16K, 16N, 16P, 16Q, 16R, 16T, 25C, 39D, 39E, 39N, 39Q, 81Y, 121P, 139D, 139H, 139R, 139Y, 177A, 188D, 191H, 191K, 192A, 192D, 192G, 192N, 192P, 192Q, 192S, 192T, 192V, 192Y, 196A, 196C, 196D, 196E, 196F, 196H, 196I, 196K, 196P, 196R, 196S, 196T, 196V, 201A, 201E, 201G, 201H, 201M, 202H, 216E, 216G, 216H, 216M, 216Q, 216R, 216S, 216T, 216Y, 221A, 221D, 221F, 221I, 221L, 221M, 221N, 221R, 221S, 221V, 221Y, 237G, 240G, 240N, 240P, 240Q, 240R, 240T, 246R, 250A, 250D, 250E, 250F, 250G, 250I, 250K, 250L, 250M, 250N, 250Q, 250R, 250S, 250W, 252K, 268A, 268D, 268E, 268G, 268H, 268K, 268N, 268P, 268Q, 268R, 268S, 274A, 274D, 274G, 2741, 274K, 274L, 274N, 274Q, 274R, 274S, 274T, 275K, 285Q, 285Y, 293K, 293R, 318A, 318F, 318G, 318I, 318K, 318L, 318M, 318R, 318S, 318T, 318V, 318Y, 319C, 319D, 319H, 3191, 319K, 319R, 319Y, 320K, 320R, 320T, 338A, 338G, 338I, 338M, 338P, 338S, 338V, 339G, 339P, 340A, 340D, 340E, 340H, 340K, 340N, 340Q, 345E, 363D, 363E, 363M, 363N, 363Q, 363S, 366Q, 370A, 370D, 370E, 370H, 370K, 370N, 370Q, 370S, 375A, 375D, 375E, 375K, 375N, 375Q, 375R, 375S, 419A, 419I, 419M, 419P, 419S, 419V, 448Y, 452N, 452Q, 452R, 452S, 471R, and 471Y; and

f) a substitution that introduces one or more of the amino acid residues selected from the group consisting of I181A, I181P, I181C, I181E, I181Y, S242A, S242E, G132A, N193Y, and E188P.

In some embodiments, the variant includes a mutation that introduces a positively charged amino acid residue one or more positions selected from the group consisting of D19, N28, E29, Q86, Q89, Q97, N224, N271, N281, D306, D318, Q319, Q358, D393, Q443, and D458, and the variant polypeptides exhibits improved cleaning performance. In particular embodiments, the improved cleaning is under North American laundry conditions, and is determined using a microswatch assay. In particular embodiments, the positively charged amino acid residue is arginine.

In some embodiments, the variant include a substitution that introduces one or more of the amino acid residues selected from the group consisting of 74A, 115L, 124K, 124R, 132A, 132C, 135A, 145A, 146A, 148A, 148N, 159A, 159C, 159D, 159E, 159F, 159G, 159H, 159K, 159L, 159N, 159R, 159S, 159T, 159V, 169A, 169L, 169M, 169Y, 179A, 181A, 181C, 181D, 181E, 181L, 181P, 181Q, 181V, 181Y, 242A, 242D, 242E, 242Q, 261L, 271A, 271V, 278A, 278H, 278K, 278N, 278R, 281A, 281L, 281M, 302D, 302M, 304D, 304E, 304M, 321A, 321H, 321Q, 321R, 333Q, 378D, 378N, 378R, 382D, 398A, 418A, 418M, 418N, 420A, 421R, 432A, 432D, 432L, 432M, 432N, 432Q, 432R, 432Y, 437D, 437G, 437H, 437L, 437M, 437Y, 446A, 446Y, 454A, 464Q, 464Y, 474A, 474E, 474K, 474L, 474M, 474N, 474P, 474Q, 474R, 474S, and 474V, and the variant has improved thermostability compared to the parental polypeptide.

In some embodiments, the variant include a substitution substitution that introduces one or more of the amino acid residues selected from the group consisting of 6I, 6N, 6Q, 6T, 6V, 14T, 16F, 25A, 25C, 25G, 25Q, 27M, 36Q, 36S, 39G, 39V, 501, SOL, 50M, 50N, 50Q, 52S, 53T, 67N, 67S, 80D, 80I, 90E, 133P, 133V, 137M, 137S, 141E, 141I, 141L, 141M, 141Q, 141R, 141S, 141V, 150E, 151I, 152G, 155S, 155Y, 168W, 173T, 188P, 193F, 193K, 193L, 193Y, 213L, 213M, 213V, 217Q, 220P, 220Q, 220R, 220S, 220V, 221I, 221S, 249E, 250F, 2501, 250M, 252L, 253Y, 254E, 254F, 254T, 254V, 255F, 255K, 255W, 257L, 257M, 257S, 257V, 258D, 258G, 258H, 258K, 258Q, 258T, 258V, 268F, 274W, 283M, 283N, 283V, 285E, 285Q, 293G, 293K, 294W, 301F, 301I, 301P, 301R, 301T, 301W, 309D, 309V, 312H, 312S, 312V, 312Y, 313G, 313H, 313I, 313L, 313S, 313V, 318T, 338A, 338C, 338G, 338M, 338T, 339K, 339T, 339V, 340A, 340M, 340Q, 340T, 343C, 3431, 343P, 343R, 343Y, 345I, 345Q, 369I, 369T, 370G, 375T, 385T, 386K, 394L, 394V, 400A, 400N, 400V, 402H, 4021, 402T, 402V, 402W, 403A, 403E, 403G, 403Q, 403R, 403T, 403V, 404C, 404E, 404G, 4041, 404V, 419A, 419C, 419M, 419T, 422E, 422G, 433A, 433H, 433I, 433K, 433L, 433M, 433V, 433Y, 442A, 442G, 442N, 442R, 442S, 442T, 442V, 442W, 442Y, 445G, 445I, 445N, 445T, 445V, 445W, 4471, 447N, 447Q, 447W, 447Y, 448C, 448F, 448G, 448H, 448I, 448N, 448Y, 450C, 450H, 450M, 450N, 450R, 450S, 450T, 450W, 455G, 455I, 455P, 455V, 463A, 463M, 463S, 463T, 463V, 463W, 465G, 465I, 465K, 465N, 465T, 465V, 469D, 469W, 469Y, 471I, 471V, 473G, 473Y, 476A, 476G, 476L, 476M, 476N, and 476T, and the variant has improved thermostability compared to the parental polypeptide.

In some embodiments, the variant include a substitution that introduces one or more of the amino acid residues selected from the group consisting of 124N, 125A, 125K, 125N, 130A, 130S, 159A, 159D, 159E, 159G, 159H, 159K, 159L, 159N, 159R, 159S, 159T, 166F, 166G, 166H, 166S, 166Y, 169L, 179A, 179P, 180A, 180D, 180H, 180K, 180L, 180N, 180T, 180V, 180Y, 181A, 181D, 181E, 181G, 181P, 181R, 181S, 181V, 187A, 187C, 187K, 187N, 187P, 187Q, 187R, 187S, 242H, 242N, 278H, 278K, 278N, 278R, 281M, 302D, 304M, 304Y, 321H, 321Q, 321R, 333Q, 432Q, 437Y, 446A, 474Q, and 474S, and the variant exhibits increased activity or expression compared to the parental polypeptide.

In some embodiments, the variant include a substitution that introduces one or more of the amino acid residues selected from the group consisting of 6A, 6D, 6E, 6H, 6I, 6K, 6L, 6M, 6N, 6P, 6Q, 6R, 6S, 6T, 6V, 6W, 6Y, 13K, 14F, 14T, 14Y, 15A, 15D, 15E, 015G, 15H, 15K, 15N, 15P, 15Q, 15R, 15S, 15T, 15W, 16A, 16E, 16G, 16H, 16K, 16N, 16P, 16Q, 16R, 16T, 25C, 39D, 39E, 39N, 39Q, 81Y, 121P, 139D, 139H, 139R, 139Y, 177A, 188D, 191H, 191K, 192A, 192D, 192G, 192N, 192P, 192Q, 192S, 192T, 192V, 192Y, 196A, 196C, 196D, 196E, 196F, 196H, 196I, 196K, 196P, 196R, 196S, 196T, 196V, 201A, 201E, 201G, 201H, 201M, 202H, 216E, 216G, 216H, 216M, 216Q, 216R, 216S, 216T, 216Y, 221A, 221D, 221F, 221I, 221L, 221M, 221N, 221R, 221S, 221V, 221Y, 237G, 240G, 240N, 240P, 240Q, 240R, 240T, 246R, 250A, 250D, 250E, 250F, 250G, 250I, 250K, 250L, 250M, 250N, 250Q, 250R, 250S, 250W, 252K, 268A, 268D, 268E, 268G, 268H, 268K, 268N, 268P, 268Q, 268R, 268S, 274A, 274D, 274G, 274I, 274K, 274L, 274N, 274Q, 274R, 274S, 274T, 275K, 285Q, 285Y, 293K, 293R, 318A, 318F, 318G, 318I, 318K, 318L, 318M, 318R, 318S, 318T, 318V, 318Y, 319C, 319D, 319H, 319I, 319K, 319R, 319Y, 320K, 320R, 320T, 338A, 338G, 338I, 338M, 338P, 338S, 338V, 339G, 339P, 340A, 340D, 340E, 340H, 340K, 340N, 340Q, 345E, 363D, 363E, 363M, 363N, 363Q, 363S, 366Q, 370A, 370D, 370E, 370H, 370K, 370N, 370Q, 370S, 375A, 375D, 375E, 375K, 375N, 375Q, 375R, 375S, 419A, 4191, 419M, 419P, 419S, 419V, 448Y, 452N, 452Q, 452R, 452S, 471R, and 471Y, and the variant exhibits increased activity or expression compared to the parental polypeptide.

In some embodiments, the variant include a substitution that introduces one or more of the amino acid residues selected from the group consisting of I181A, I181P, I181C, I181E, I181Y, S242A, S242E, G132A, N193Y, and E188P, and the variant exhibits increased viscosity reduction in a starch liquefaction assay compared to the parental polypeptide.

In some embodiments, a variant α-amylase polypeptide is provided, comprising an amino acid sequence derived from a parental α-amylase polypeptide, and having a combination of three or more mutations at positions selected from the group consisting of 5, 6, 13, 14, 15, 16, 18, 20, 25, 27, 29, 36, 39, 50, 52, 53, 54, 67, 71, 73, 75, 77, 80, 81, 83, 85, 90, 92, 107, 111, 113, 114, 120, 121, 126, 128, 131, 133, 137, 138, 139, 141, 143, 147, 149, 150, 151, 152, 155, 160, 165, 168, 172, 173, 177, 188, 191, 192, 193, 196, 200, 201, 202, 213, 216, 217, 220, 221, 227, 232, 235, 237, 238, 240, 246, 249, 250, 252, 253, 254, 255, 257, 258, 268, 272, 274, 275, 279, 283, 285, 293, 294, 297, 300, 301, 306, 309, 312, 313, 317, 318, 319, 320, 338, 339, 340, 343, 345, 363, 366, 369, 370, 375, 379, 381, 385, 386, 391, 392, 393, 394, 400, 402, 403, 404, 406, 407, 410, 413, 414, 416, 419, 422, 427, 433, 436, 439, 442, 445, 447, 448, 450, 452, 455, 463, 465, 469, 471, 473, and 476, wherein the polypeptides has α-amylase activity, and where each of the at least three or more mutations introduces an amino acid residue that differs from that in the parental polypeptide. In particular embodiments, the number of mutations is 4, 5, 6, 7, 8, 9, 10, or more.

In some embodiments, where the mutation is not already at position 242, the mutation is present in combination with the substitutions S242A, S242E, S242Q, S242F, S242H, or S242N. In particular embodiments, the substitution is S242Q. In some embodiments, where the mutation is not already at position 179 or 180, the mutation is present in combination with a deletion at positions 179 and 180. In some embodiments, where the mutation is not already at position 349 or 428, the mutation is present in combination with a substitution of a cysteine at one or more of these amino acids.

In some embodiments, where the mutation is not already at one of the following positions, the mutation is present in combination with a substitution at position P17, D19, T21, N28, S51, G72, V74, A82, Q86, Q89, A93, G95, Q97, W115, D117, P123, S124, D125, N127, I130, G132, Q135, P145, G146, G148, S153, Y159, W166, S169, K171, W187, P209, N224, S242, G256, D269, N271, T278, N281, G302, A304, R308, T321, Q358, P378, S382, K383, T398, H405, T417, E418, P420, G421, P432, W437, G446, G454, S457, T459, T461, S464, G474, or R483.

In some embodiments, where the mutation is not already at one of the following positions, the mutation is present in combination with a substitution at position M8, M9, M15, M96, V128, A111, H133, W138, T149, M197, N188, M200, M206, A209, A210, M284, M307, M311, M316, H405, T412, M438, N193F, and V416G.

In some embodiments, the parental polypeptide has at least 80%, at least 85%, at least 90%, or even at least 95% amino acid sequence identity to the polypeptide of SEQ ID NO: 1.

In some embodiments, the parental polypeptide has at least 80%, at least 85%, at least 90%, or even at least 95% amino acid sequence identity to the polypeptide of SEQ ID NO: 2.

In some embodiments, the parental polypeptide has at least 80%, at least 85%, at least 90%, or even at least 95% amino acid sequence identity to a polypeptide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 15, and SEQ ID NO: 16. In some embodiments, the parental polypeptide includes a truncation of the C-terminal amino acid residues. In particular embodiments, the truncation is of the C-terminal 29 amino acid residues.

In some embodiments, the variant polypeptide does not include a mutations at either position 106 or 199, or both.

In some embodiments, one or more mutations can be added or deleted from a list of mutations without departing from the description. Relatedly, any one or more mutations that appear in the context of a list of mutations can be combined as a subset of mutations.

In another aspect, a composition comprising one or more of the aforementioned variant α-amylases is provided. In particular embodiments, the composition is a cleaning composition, such as a laundry detergent, a dishwashing detergent, a hard-surface-cleaning composition, or the like. The composition may include a detergent.

In another aspect, a method for hydrolyzing a soluble starch substrate using an α-amylase variant is provided. In some embodiments, the variant includes a substitution that introduces one or more of the amino acid residues selected from the group consisting of I181A, I181P, I181C, I181E, I181Y, S242A, S242E, S242Q, G132A, N193Y, and E188P.

In some embodiments, the variant α-amylase is used incombination with a phytic acid hydrolyzing enzyme, wherein the ratio of α-amylase activity (in α-amylase units) to phytic acid activity (in phytase units), i.e., AAU:FTU, is from about 1:15 to about 15:1, and preferably from 1:10 to about 10:1. In particular embodiments, the ratio of AAU:FTU is from 1:4 to 3:1, or even 1:1.

In a further aspect, a method for liquefying starch in a slurry is provided, involving a substrate that includes plant material such as granular starch from either a dry or wet milling process, the method comprising a primary and/or secondary liquefaction step, involving adding to the slurry in the primary and/or secondary liquefaction step, in any order, a combination of at least one phytic acid hydrolyzing enzyme and at least one variant α-amylase, either simultaneously or separately. The method can further comprise saccharifying the liquefied starch to obtain fermentable sugars; and recovering the fermentable sugars. In some embodiments, the method further comprises fermenting the fermentable sugars under suitable fermentation conditions to obtain end-products such as alcohol. In some embodiments the enzyme composition contains at least one variant α-amylase and a phytase. In some embodiments, the enzyme composition is in blended form.

In a further aspect, a method for fermenting a starch substrate is provided, the method comprising adding in any order a combination of a variant α-amylase and a phytase in a single or split dose. In another aspect, the treated starch substrate is fermented to ethanol.

In a further aspect, a starch conversion process and/or an ethanol fermentation process is provided that does not require addition of acid or alkali to adjust the pH. One embodiment relates to a pH adjustment free liquefaction step, wherein the pH of the liquefaction is in the range of pH 4.5 to 5.4 and acid neutralizing chemicals are not added to the liquefaction process step. In another embodiment, the pH of the liquefaction is in the range of pH 4.8 to 5.8 and acid neutralizing chemicals are not added to the liquefaction process step.

In another aspect, a method of obtaining a fermentable substrate is provided, involving contacting a slurry of milled grain containing granular starch with a phytic acid hydrolyzing enzyme at a temperature 0-30° C. less than the starch gelatinization temperature, contacting the slurry with a variant α-amylase, raising the temperature above the gelatinization temperature for the granular starch to allow gelatinization of the starch, and hydrolyzing the gelatinized starch by contacting the gelatinized starch with the α-amylase for a time sufficient to hydrolyze the starch, and obtaining a fermentable substrate. The phytic acid hydrolyzing enzyme can be a bacterial or fungal phytase. The fungal phytase can be an Aspergillus phytase or a Buttiauxella phytase. In some embodiments, the bacterial phytase is from Escherichia coli.

In another aspect, the a process for producing a fermentable sugar is provided, comprising (a) mixing milled starch-containing material with water and thin stillage, wherein the thin stillage is in the range of 10 to 70% v/v and obtaining a slurry comprising starch and having a dry solids (ds) content of 20 to 50% w/w, (b) treating the slurry with a phytase prior to or simultaneously with liquefying the starch, (c) liquefying the starch, (d) adding a variant α-amylase to the starch either during step (b) and/or simultaneously with the liquefying step, and (e) saccharifying the liquefied starch to obtain fermentable sugars, wherein the pH is not adjusted during any of the steps (a), (b), (c), (d), or (e). In some embodiments, the fermentable sugar is recovered and purified or isomerized. In other embodiments, the phytase is added prior to the liquefaction step. In some embodiments, the α-amylase is added with the phytase. In yet further embodiments, a second α-amylase dose is added during the liquefaction step.

In a further aspect, a process of producing alcohol from the starch-containing material, is provided, comprising liquefying and saccharifying the liquefied starch as disclosed above to obtain fermentable sugars and further fermenting the fermentable sugars under suitable fermentation conditions using a fermenting microorganism to obtain alcohol. In some embodiments, the saccharification and fermentation steps are simultaneous. In some embodiments, the alcohol is ethanol.

In another aspect, DNA constructs, including expression vectors, that encode variants α-amylases are provided, along with methods of expressing and using the variant α-amylases, alone or in combination with other α-amylolytic enzymes, e.g., in various industrial processes, such as starch liquefaction and cleaning.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an alignment of the amino acid sequences of several AmyS-related α-amylases.

FIG. 2 shows the pHPLT-AmyS plasmid.

FIG. 3 shows percent residual activity of S242 library variants after heat stress at 95° C. for 30 minutes. Variant positions P, S, W, and Y are missing and are replaced by wild type AmyS (SPEZYME® Xtra—labeled “Z”). Lines indicate 2× and 3× above the standard deviation of the percent residual activity of the wildtype enzyme. S242A and S242Q clearly show higher residual activities than the wild type.

FIGS. 4A-4I shows pairwise alignments of the amino acid sequences shown in FIG. 1.

FIG. 5 shows the thermal melting curves and the melting points for the wild type and amylase variants without added calcium.

FIG. 6 shows the thermal melting curves and the melting points for the wild type and amylase variants with 2 mM calcium.

FIG. 7 shows the activity profile of SPEZYME® Xtra and two variants relative to Liquozyme SC for three time points.

FIG. 8 shows the activity profile of four variants relative to the S242Q variant for three time points.

FIG. 9 shows the viscosity reduction of corn flour due to the action of the α-amylases LIQUOZYME® SC or SPEZYME® Xtra at a 30 μg dose.

FIG. 10 shows the viscosity reduction of corn flour due to the action of the α-amylases LIQUOZYME® SC or SPEZYME® Xtra, or one of two variants (S242A and S242Q) at a 30 μg dose.

FIG. 11 shows the viscosity reduction of corn flour due to the action of the α-amylase LIQUOZYME® SC or SPEZYME® Xtra, or one of two variants (S242A and S242Q) at a 20 μg dose.

FIG. 12 shows the DE progression of whole ground corn treated with LIQUOZYME® SC, SPEZYME® Xtra, or one of two variants (S242A and S242Q) over time (0, 30, 60, and 90 minutes).

FIG. 13 shows the viscosity post-jet of whole ground corn treated with LIQUOZYME® SC, SPEZYME® Xtra, or one of two variants (S242A and S242Q) over time (0, 30, 60, and 90 minutes).

FIG. 14 shows the DE progression of whole ground corn treated with phytase and an amylase (SPEZYME® Xtra or S242Q variant) over time (0, 30, 60 and 90 minutes). MAXALIQ® is a phytase/amylase blend available from Genencor, a Danisco Division. Reference is made to Example 8.

FIG. 15 shows the viscosity post-jet of whole ground corn treated with phytase and an amylase (SPEZYME® Xtra or S242Q variant) over time (0, 30, 60, and 90 minutes).

FIG. 16 shows the DE progression of whole ground corn treated with of the S242Q variant and phytase. Reference is made to Example 9.

FIG. 17 shows the viscosity post-jet of whole ground corn treated with of the S242Q variant and phytase. Reference is made to Example 9.

FIG. 18 shows the effect of phytase treatment of whole ground corn on the increase in the thermostability and low pH stability of the S242Q variant and reference is made to Example 9.

FIG. 19 shows the effect of phytase addition during primary liquefaction of whole ground corn on the viscosity reduction after jet cooking and reference is made to Example 9.

FIG. 20 shows a comparison of sulfate and phytic acid content in DDGS: 1) from a conventional process, and 2) from the process with no pH adjustment. Reference is made to Example 10.

FIG. 21 is a graph showing the rate of DE progression and the percent phytic acid reduction as IP6.

FIG. 22 is a graph showing the effect of the S242Q variant α-amylase on DE progression under conventional processing conditions. Reference is made to Example 8.

FIG. 23 is a graph depicting the performance of S242Q and its variants in the rice starch microswatch assay as a function of charge under North American laundry conditions. The conditions were TIDE® 2× at 20° C. Reference is made to Example 16.

FIG. 24 is a graph depicting the performance of another α-amylase (i.e., truncated Bacillus sp. TS-23 amylase with the charge mutations in the rice starch microswatch assay under Western European laundry conditions. The conditions were PERSIL® at 40° C. Reference is made to Example 16.

FIG. 25 is a graph depicting the performance of S242Q and its variants in the BODIPY-starch assay as a function of charge. Reference is made to Example 16.

FIG. 26 A is a graph depicting the relative BODIPY-starch hydrolysis as a function of relative shake tube expression (i.e., relative BODIPY-starch hydrolysis vs. relative shake tube expression). FIG. 26B is a graph depicting the relative microswatch-starch hydrolysis as a function of relative shake tube expression (i.e., relative microswatch-starch hydrolysis vs. relative shake tube expression). Reference is made to Example 19.

FIG. 27A is a graph depicting the relative shake tube expression as a function of charge. FIG. 27B is a graph depicting the relative BODIPY-starch hydrolysis as a function of charge. Reference is made to Example 19.

FIG. 28A is a graph depicting the relative shake tube expression as a function of charge. FIG. 28B is a graph depicting the relative microswatch cleaning activity as a function of charge. Reference is made to Example 19.

FIG. 29 is a graph depicting the final viscosity after corn starch liquefaction using 1st AmyS Ladder 30% DS, pH 5.8, enzyme dose 30 mg. For the +6 variant final viscosity is so high and cannot be measured (instrument overload). Reference is made to Example 16.

FIG. 30 is a graph depicting the thermal stability of the first AmyS charge ladder as a function of charge change relative to wild type. Experiment performed using standard amylase thermal stability assay. Reference is made to Example 17.

FIG. 31A is a graph depicting the rice starch cleaning activity of first AmyS charge ladder as a function of pH. pH 3.0-4.25 is 200 mM Na formate+0.01% Tween-80. pH 4.25-5.5 is 200 mM Na acetate+0.01% Tween-80. The data are fit to titration curves, each with a single pKa value. Reference is made to Example 21.

FIG. 31B is a graph depicting the effect of charge mutations on apparent pKa for AmyS catalysis (first charge ladder). Reference is made to Example 21.

FIG. 32A shows the viscosity reduction of corn flour by AmyS variants compared to SPEZYME® Xtra.

FIG. 32B shows viscosity reduction of corn fluor by AmyS N193Y.

FIG. 32C shows the effect of phytase addition on viscosity reduction by AmyS N193Y.

BRIEF DESCRIPTION OF THE SEQUENCES

The following amino acid and nucleotide sequences are referred to herein.

SEQ ID NO: 1 (full-length, wild-type AmyS) AAPFNGTMMQ YFEWYLPDDG TLWTKVANEA NNLSSLGITA LWLPPAYKGT SRSDVGYGVY DLYDLGEFNQ KGTVRTKYGT KAQYLQAIQA AHAAGMQVYA DVVFDHKGGA DGTEWVDAVE VNPSDRNQEI SGTYQIQAWT KFDFPGRGNT YSSFKWRWYH FDGVDWDESR KLSRIYKFRG IGKAWDWEVD TENGNYDYLM YADLDMDHPE VVTELKNWGK WYVNTTNIDG FRLDAVKHIK FSFFPDWLSY VRSQTGKPLF TVGEYWSYDI NKLHNYITKT NGTMSLFDAP LHNKFYTASK SGGAFDMRTL MTNTLMKDQP TLAVTFVDNH DTEPGQALQS WVDPWFKPLA YAFILTRQEG YPCVFYGDYY GIPQYNIPSL KSKIDPLLIA RRDYAYGTQH DYLDHSDIIG WTREGVTEKP GSGLAALITD GPGGSKWMYV GKQHAGKVFY DLTGNRSDTV TINSDGWGEF KVNGGSVSVW VPRKTTVSTI ARPITTRPWT GEFVRWTEPR LVAWP SEQ ID NO: 2 (truncated, wild-type AmyS; SPEZYME ® Xtra) AAPFNGTMMQ YFEWYLPDDG TLWTKVANEA NNLSSLGITA LWLPPAYKGT SRSDVGYGVY DLYDLGEFNQ KGTVRTKYGT KAQYLQAIQA AHAAGMQVYA DVVFDHKGGA DGTEWVDAVE VNPSDRNQEI SGTYQIQAWT KFDFPGRGNT YSSFKWRWYH FDGVDWDESR KLSRIYKFRG IGKAWDWEVD TENGNYDYLM YADLDMDHPE VVTELKNWGK WYVNTTNIDG FRLDAVKHIK FSFFPDWLSY VRSQTGKPLF TVGEYWSYDI NKLHNYITKT NGTMSLFDAP LHNKFYTASK SGGAFDMRTL MTNTLMKDQP TLAVTFVDNH DTEPGQALQS WVDPWFKPLA YAFILTRQEG YPCVFYGDYY GIPQYNIPSL KSKIDPLLIA RRDYAYGTQH DYLDHSDIIG WTREGVTEKP GSGLAALITD GPGGSKWMYV GKQHAGKVFY DLTGNRSDTV TINSDGWGEF KVNGGSVSVW VPRKTT SEQ ID NO: 3 (full-length, S242A AmyS) AAPFNGTMMQ YFEWYLPDDG TLWTKVANEA NNLSSLGITA LWLPPAYKGT SRSDVGYGVY DLYDLGEFNQ KGTVRTKYGT KAQYLQAIQA AHAAGMQVYA DVVFDHKGGA DGTEWVDAVE VNPSDRNQEI SGTYQIQAWT KFDFPGRGNT YSSFKWRWYH FDGVDWDESR KLSRIYKFRG IGKAWDWEVD TENGNYDYLM YADLDMDHPE VVTELKNWGK WYVNTTNIDG FRLDAVKHIK FAFFPDWLSY VRSQTGKPLF TVGEYWSYDI NKLHNYITKT NGTMSLFDAP LHNKFYTASK SGGAFDMRTL MTNTLMKDQP TLAVTFVDNH DTEPGQALQS WVDPWFKPLA YAFILTRQEG YPCVFYGDYY GIPQYNIPSL KSKIDPLLIA RRDYAYGTQH DYLDHSDIIG WTREGVTEKP GSGLAALITD GPGGSKWMYV GKQHAGKVFY DLTGNRSDTV TINSDGWGEF KVNGGSVSVW VPRKTTVSTI ARPITTRPWT GEFVRWTEPR LVAWP SEQ ID NO: 4 (full-length, S242Q AmyS) AAPFNGTMMQ YFEWYLPDDG TLWTKVANEA NNLSSLGITA LWLPPAYKGT SRSDVGYGVY DLYDLGEFNQ KGTVRTKYGT KAQYLQAIQA AHAAGMQVYA DVVFDHKGGA DGTEWVDAVE VNPSDRNQEI SGTYQIQAWT KFDFPGRGNT YSSFKWRWYH FDGVDWDESR KLSRIYKFRG IGKAWDWEVD TENGNYDYLM YADLDMDHPE VVTELKNWGK WYVNTTNIDG FRLDAVKHIK FQFFPDWLSY VRSQTGKPLF TVGEYWSYDI NKLHNYITKT NGTMSLFDAP LHNKFYTASK SGGAFDMRTL MTNTLMKDQP TLAVTFVDNH DTEPGQALQS WVDPWFKPLA YAFILTRQEG YPCVFYGDYY GIPQYNIPSL KSKIDPLLIA RRDYAYGTQH DYLDHSDIIG WTREGVTEKP GSGLAALITD GPGGSKWMYV GKQHAGKVFY DLTGNRSDTV TINSDGWGEF KVNGGSVSVW VPRKTTVSTI ARPITTRPWT GEFVRWTEPR LVAWP SEQ ID NO: 5 (full-length, S242E AmyS) AAPFNGTMMQ YFEWYLPDDG TLWTKVANEA NNLSSLGITA LWLPPAYKGT SRSDVGYGVY DLYDLGEFNQ KGTVRTKYGT KAQYLQAIQA AHAAGMQVYA DVVFDHKGGA DGTEWVDAVE VNPSDRNQEI SGTYQIQAWT KFDFPGRGNT YSSFKWRWYH FDGVDWDESR KLSRIYKFRG IGKAWDWEVD TENGNYDYLM YADLDMDHPE VVTELKNWGK WYVNTTNIDG FRLDAVKHIK FEFFPDWLSY VRSQTGKPLF TVGEYWSYDI NKLHNYITKT NGTMSLFDAP LHNKFYTASK SGGAFDMRTL MTNTLMKDQP TLAVTFVDNH DTEPGQALQS WVDPWFKPLA YAFILTRQEG YPCVFYGDYY GIPQYNIPSL KSKIDPLLIA RRDYAYGTQH DYLDHSDIIG WTREGVTEKP GSGLAALITD GPGGSKWMYV GKQHAGKVFY DLTGNRSDTV TINSDGWGEF KVNGGSVSVW VPRKTTVSTI ARPITTRPWT GEFVRWTEPR LVAWP SEQ ID NO: 6 (Yamane 707) HHNGTNGTMMQYFEWYLPNDGNHWNRLNSDASNLKSKGITAVWIPPAWKGASQNDVGYGAYDLYD LGEFNQKGTVRTKYGTRSQLQAAVTSLKNNGIQVYGDVVMNHKGGADATEMVRAVEVNPNNRNQE VTGEYTIEAWTRFDFPGRGNTHSSFKWRWYHFDGVDWDQSRRLNNRIYKFRGHGKAWDWEVDTEN GNYDYLMYADIDMDHPEVVNELRNWGVWYTNTLGLDGFRIDAVKHIKYSFTRDWINHVRSATGKN MFAVAEFWKNDLGAIENYLQKTNWNHSVFDVPLHYNLYNASKSGGNYDMRNIFNGTVVQRHPSHA VTFVDNHDSQPEEALESFVEEWFKPLAYALTLTREQGYPSVFYGDYYGIPTHGVPAMRSKIDPIL EARQKYAYGKQNDYLDHHNIIGWTREGNTAHPNSGLATIMSDGAGGSKWMFVGRNKAGQVWSDIT GNRTGTVTINADGWGNFSVNGGSVSIWVNK SEQ ID NO: 7 (wild-type AmyL; LAT) ANLNGTLMQYFEWYMPNDGQHWKRLQNDSAYLAEHGITAVWIPPAYKGTSQADVGYGAYDLYDLG EFHQKGTVRTKYGTKGELQSAIKSLHSRDINVYGDVVINHKGGADATEDVTAVEVDPADRNRVIS GEHLIKAWTHFHFPGRGSTYSDFKWHWYHFDGTDWDESRKLNRIYKFQGKAWDWEVSNENGNYDY LMYADIDYDHPDVAAEIKRWGTWYANELQLDGFRLDAVKHIKFSFLRDWVNHVREKTGKEMFTVA EYWQNDLGALENYLNKTNFNHSVFDVPLHYQFHAASTQGGGYDMRKLLNGTVVSKHPLKSVTFVD NHDTQPGQSLESTVQTWFKPLAYAFILTRESGYPQVFYGDMYGTKGDSQREIPALKHKIEPILKA RKQYAYGAQHDYFDHHDIVGWTREGDSSVANSGLAALITDGPGGAKRMYVGRQNAGETWHDITGN RSEPVVINSEGWGEFHVNGGSVSIYVQR SEQ ID NO: 8 (wild-type AmyL; Termamyl) ANLNGTLMQYFEWYMPNDGQHWRRLQNDSAYLAEHGITAVWIPPAYKGTSQADVGYGAYDLYDLG EFHQKGTVRTKYGTKGELQSAIKSLHSRDINVYGDVVINHKGGADATEDVTAVEVDPADRNRVIS GEHLIKAWTHFHFPGRGSTYSDFKWHWYHFDGTDWDESRKLNRIYKFQGKAWDWEVSNENGNYDY LMYADIDYDHPDVAAEIKRWGTWYANELQLDGFRLDAVKHIKFSFLRDWVNHVREKTGKEMFTVA EYWQNDLGALENYLNKTNFNHSVFDVPLHYQFHAASTQGGGYDMRKLLNGTVVSKHPLKSVTFVD NHDTQPGQSLESTVQTWFKPLAYAFILTRESGYPQVFYGDMYGTKGDSQREIPALKHKIEPILKA RKQYAYGAQHDYFDHHDIVGWTREGDSSVANSGLAALITDGPGGAKRMYVGRQNAGETWHDITGN RSEPVVINSEGWGEFHVNGGSVSIYVQR SEQ ID NO: 9 (B. amyloliquefaciens amylase) VNGTLMQYFEWYTPNDGQHWKRLQNDAEHLSDIGITAVWIPPAYKGLSQSDNGYGPYDLYDLGEF QQKGTVRTKYGTKSELQDAIGSLHSRNVQVYGDVVLNHKAGADATEDVTAVEVNPANRNQETSEE YQIKAWTDFRFPGRGNTYSDFKWHWYHFDGADWDESRKISRIFKFRGEGKAWDWEVSSENGNYDY LMYADVDYDHPDVVAETKKWGIWYANELSLDGFRIDAAKHIKFSFLRDWVQAVRQATGKEMFTVA EYWQNNAGKLENYLNKTSFNQSVFDVPLHFNLQAASSQGGGYDMRRLLDGTVVSRHPEKAVTFVE NHDTQPGQSLESTVQTWFKPLAYAFILTRESGYPQVFYGDMYGTKGTSPKEIPSLKDNIEPILKA RKEYAYGPQHDYIDHPDVIGWTREGDSSAAKSGLAALITDGPGGSKRMYAGLKNAGETWYDITGN RSDTVKIGSDGWGEFHVNDGSVSIYVQK SEQ ID NO: 10 (STAINZYME ™) HHNGTNGTMM QYFEWYLPND GNHWNRLRSD ASNLKDKGIS AVWIPPAWKG ASQNDVGYGA YDLYDLGEFN QKGTIRTKYG TRNQLQAAVN ALKSNGIQVY GDVVMNHKGG ADATEMVRAV EVNPNNRNQE VSGEYTIEAW TKFDFPGRGN THSNFKWRWY HFDGVDWDQS RKLNNRIYKF RGDGKGWDWE VDTENGNYDY LMYADIDMDH PEVVNELRNW GVWYTNTLGL DGFRIDAVKH IKYSFTRDWI NHVRSATGKN MFAVAEFWKN DLGAIENYLN KTNWNHSVFD VPLHYNLYNA SKSGGNYDMR QIFNGTVVQR HPMHAVTFVD NHDSQPEEAL ESFVEEWFKP LAYALTLTRE QGYPSVFYGD YYGIPTHGVP AMKSKIDPIL EARQKYAYGR QNDYLDHHNI IGWTREGNTA HPNSGLATIM SDGAGGNKWM FVGRNKAGQV WTDITGNRAG TVTINADGWG NFSVNGGSVS IWVNK SEQ ID NO: 11 (NATALASE ™) HHNGTNGTMMQYFEWHLPNDGNHWNRLRDDASNLRNRGITAIWIPPAWKGTSQNDVGYGAYDLYD LGEFNQKGTVRTKYGTRSQLESAIHALKNNGVQVYGDVVMNHKGGADATENVLAVEVNPNNRNQE ISGDYTIEAWTKFDFPGRGNTYSDFKWRWYHFDGVDWDQSRQFQNRIYKFRGDGKAWDWEVDSEN GNYDYLMYADVDMDHPEVVNELRRWGEWYTNTLNLDGFRIDAVKHIKYSFTRDWLTHVRNATGKE MFAVAEFWKNDLGALENYLNKTNWNHSVFDVPLHYNLYNASNSGGNYDMAKLLNGTVVQKHPMHA VTFVDNHDSQPGESLESFVQEWFKPLAYALILTREQGYPSVFYGDYYGIPTHSVPAMKAKIDPIL EARQNFAYGTQHDYFDHHNIIGWTREGNTTHPNSGLATIMSDGPGGEKWMYVGQNKAGQVWHDIT GNKPGTVTINADGWANFSVNGGSVSIWVKR SEQ ID NO: 12 (KAO KSM 1378) HHNGTNGTMMQYFEWHLPNDGNHWNRLRDDAANLKSKGITAVWIPPAWKGTSQNDVGYGAYDLYD LGEFNQKGTVRTKYGTRSQLQGAVTSLKNNGIQVYGDVVMNHKGGADGTEMVNAVEVNRSNRNQE ISGEYTIEAWTKFDFPGRGNTHSNFKWRWYHFDGTDWDQSRQLQNKIYKFRGTGKAWDWEVDIEN GNYDYLMYADIDMDHPEVINELRNWGVWYTNTLNLDGFRIDAVKHIKYSYTRDWLTHVRNTTGKP MFAVAEFWKNDLAAIENYLNKTSWNHSVFDVPLHYNLYNASNSGGYFDMRNILNGSVVQKHPIHA VTFVDNHDSQPGEALESFVQSWFKPLAYALILTREQGYPSVFYGDYYGIPTHGVPSMKSKIDPLL QARQTYAYGTQHDYFDHHDIIGWTREGDSSHPNSGLATIMSDGPGGNKWMYVGKHKAGQVWRDIT GNRSGTVTINADGWGNFTVNGGAVSVWVKQ SEQ ID NO: 13 (KAO KSM K38) DGLNGTMMQYYEWHLENDGQHWNRLHDDAAALSDAGITAIWIPPAYKGNSQADVGYGAYDLYDLG EFNQKGTVRTKYGTKAQLERAIGSLKSNDINVYGDVVMNHKMGADFTEAVQAVQVNPTNRWQDIS GAYTIDAWTGFDFSGRNNAYSDFKWRWFHFNGVDWDQRYQENHIFRFANTNWNWRVDEENGNYDY LLGSNIDFSHPEVQDELKDWGSWFTDELDLDGYRLDAIKHIPFWYTSDWVRHQRNEADQDLFVVG EYWKDDVGALEFYLDEMNWEMSLFDVPLNYNFYRASQQGGSYDMRNILRGSLVEAHPMHAVTFVD NHDTQPGESLESWVADWFKPLAYATILTREGGYPNVFYGDYYGIPNDNISAKKDMIDELLDARQN YAYGTQHDYFDHWDVVGWTREGSSSRPNSGLATIMSNGPGGSKWMYVGRQNAGQTWTDLTGNNGA SVTINGDGWGEFFTNGGSVSVYVNQ SEQ ID NO: 14 (KAO KSM K36) DGLNGTMMQYYEWHLENDGQHWNRLHDDAEALSNAGITAIWIPPAYKGNSQADVGYGAYDLYDLG EFNQKGTVRTKYGTKAQLERAIGSLKSNDINVYGDVVMNHKLGADFTEAVQAVQVNPSNRWQDIS GVYTIDAWTGFDFPGRNNAYSDFKWRWFHFNGVDWDQRYQENHLFRFANTNWNWRVDEENGNYDY LLGSNIDFSHPEVQEELKDWGSWFTDELDLDGYRLDAIKHIPFWYTSDWVRHQRSEADQDLFVVG EYWKDDVGALEFYLDEMNWEMSLFDVPLNYNFYRASKQGGSYDMRNILRGSLVEAHPIHAVTFVD NHDTQPGESLESWVADWFKPLAYATILTREGGYPNVFYGDYYGIPNDNISAKKDMIDELLDARQN YAYGTQHDYFDHWDIVGWTREGTSSRPNSGLATIMSNGPGGSKWMYVGQQHAGQTWTDLTGNHAA SVTINGDGWGEFFTNGGSVSVYVNQ SEQ ID NO: 15 (LIQUIZYME ® SC) AAPFNGTMMQYFEWYLPDDGTLWTKVANEANNLSSLGITALWLPPAYKGTSRSDVGYGVYDLYDL GEFNQKGTVRTKYGTKAQYLQAIQAAHAAGMQVYADVVFDHKGGADGTEWVDAVEVNPSDRNQEI SGTYQIQAWTKFDFPGRGNTYSSFKWRWYHFDGVDWDESRKLSRIYKFRGKAWDWEVDTEFGNYD YLMYADLDMDHPEVVTELKNWGKWYVNTTNIDGFRLDAVKHIKFSFFPDWLSYVRSQTGKPLFTV GEYWSYDINKLHNYITKTNGTMSLFDAPLHNKFYTASKSGGAFDMRTLMTNTLMKDQPTLAVTFV DNHDTEPGQALQSWVDPWFKPLAYAFILTRQEGYPCVFYGDYYGIPQYNIPSLKSKIDPLLIARR DYAYGTQHDYLDHSDIIGWTREGGTEKPGSGLAALITDGPGGSKWMYVGKQHAGKVFYDLTGNRS DTVTINSDGWGEFKVNGGSVSVWVPRKTTVS SEQ ID NO: 16 (SPEZYME ® Ethyl) AAPFNGTMMQYFEWYLPDDGTLWTKVANEANNLSSLGITALWLPPAYKGTSRSDVGYGVYDLYDL GEFNQKGTVRTKYGTKAQYLQAIQAAHAAGMQVYADVVFDHKGGADGTEWVDAVEVNPSDRNQEI SGTYQIQAWTKFDFPGRGNTYSSFKWRWYHFDGVDWDESRKLSRIYKFIGKAWDWEVDTENGNYD YLMYADLDMDHPEVVTELKNWGKWYVNTTNIDGFRLDAVKHIKFSFFPDWLSYVRSQTGKPLFTV GEYWSYDINKLHNYITKTNGTMSLFDAPLHNKFYTASKSGGAFDMRTLMTNTLMKDQPTLAVTFV DNHDTEPGQALQSWVDPWFKPLAYAFILTRQEGYPCVFYGDYYGIPQYNIPSLKSKIDPLLIARR DYAYGTQHDYLDHSDIIGWTREGVTEKPGSGLAALITDGPGGSKWMYVGKQHAGKVFYDLTGNRS DTVTINSDGWGEFKVNGGSVSVWVPRKTT SEQ ID NO: 17 (primer S242 F) 5′-[Phos]GTCAAGCATATTAAGTTCNNSTTTTTTCCTGATTGGTTG-3′ SEQ ID NO: 18 (primer S242 R) 5′-[Phos]CAACCAATCAGGAAAAAASNNGAACTTAATATGCTTGAC-3′ SEQ ID NO: 19 (BP17 phytase) NDTPASGYQV EKVVILSRHG VRAPTKMTQT MRDVTPNTWP EWPVKLGYIT PRGEHLISLM GGFYRQKFQQ QGILSQGSCP TPNSIYVWAD VDQRTLKTGE AFLAGLAPQC GLTIHHQQNL EKADPLFHPV KAGTCSMDKT QVQQAVEKEA QTPIDNLNQH YIPFLALMNT TLNFSTSAWC QKHSADKSCD LGLSMPSKLS IKDNGNKVAL DGAIGLSSTL AEIFLLEYAQ GMPQAAWGNI HSEQEWASLL KLHNVQFDLM ARTPYIARHN GTPLLQAISN ALNPNATESK LPDISPDNKI LFIAGHDTNI ANIAGMLNMR WTLPGQPDNT PPGGALVFER LADKSGKQYV SVSMVYQTLE QLRSQTPLSL NQPAGSVQLK IPGCNDQTAE GYCPLSTFTR VVSQSVEPGC QLQ SEQ ID NO: 20 (coding sequence for the LAT signal peptide) atgaaacaac aaaaacggct ttacgcccga ttgctgacgc tgttatttgc gctcatcttc ttgctgcctc attctgcagc ttcagca SEQ ID NO: 21 (LAT signal peptide) MKQQKRLYAR LLTLLFALIF LLPHSAASA SEQ ID NO: 22 (truncated S242Q AmyS) AAPFNGTMMQ YFEWYLPDDG TLWTKVANEA NNLSSLGITA LWLPPAYKGT SRSDVGYGVY DLYDLGEFNQ KGTVRTKYGT KAQYLQAIQA AHAAGMQVYA DVVFDHKGGA DGTEWVDAVE VNPSDRNQEI SGTYQIQAWT KFDFPGRGNT YSSFKWRWYH FDGVDWDESR KLSRIYKFRG IGKAWDWEVD TENGNYDYLM YADLDMDHPE VVTELKNWGK WYVNTTNIDG FRLDAVKHIK FQFFPDWLSY VRSQTGKPLF TVGEYWSYDI NKLHNYITKT NGTMSLFDAP LHNKFYTASK SGGAFDMRTL MTNTLMKDQP TLAVTFVDNH DTEPGQALQS WVDPWFKPLA YAFILTRQEG YPCVFYGDYY GIPQYNIPSL KSKIDPLLIA RRDYAYGTQH DYLDHSDIIG WTREGVTEKP GSGLAALITD GPGGSKWMYV GKQHAGKVFY DLTGNRSDTV TINSDGWGEF KVNGGSVSVW VPRKTT SEQ ID NO: 23 (coding sequence for mature AmyS) gccgcaccgt ttaacggtac catgatgcag tattttgaat ggtacttgcc ggatgatggc acgttatgga ccaaagtggc caatgaagcc aacaacttat ccagccttgg catcaccgct ctttggctgc cgcccgctta caaaggaaca agccgcagcg acgtagggta cggagtatac gacttgtatg acctcggcga attcaatcaa aaagggaccg tccgcacaaa atatggaaca aaagctcaat atcttcaagc cattcaagcc gcccacgccg ctggaatgca agtgtacgcc gatgtcgtgt tcgaccataa aggcggcgct gacggcacgg aatgggtgga cgccgtcgaa gtcaatccgt ccgaccgcaa ccaagaaatc tcgggcacct atcaaatcca agcatggacg aaatttgatt ttcccgggcg gggcaacacc tactccagct ttaagtggcg ctggtaccat tttgacggcg ttgactggga cgaaagccga aaattaagcc gcatttacaa attccgcggc atcggcaaag cgtgggattg ggaagtagac acggaaaacg gaaactatga ctacttaatg tatgccgacc ttgatatgga tcatcccgaa gtcgtgaccg agctgaaaaa ctgggggaaa tggtatgtca acacaacgaa cattgatggg ttccggcttg atgccgtcaa gcatattaag ttcagttttt ttcctgattg gttgtcgtat gtgcgttctc agactggcaa gccgctattt accgtcgggg aatattggag ctatgacatc aacaagttgc acaattacat tacgaaaaca aacggaacga tgtctttgtt tgatgccccg ttacacaaca aattttatac cgcttccaaa tcagggggcg catttgatat gcgcacgtta atgaccaata ctctcatgaa agatcaaccg acattggccg tcaccttcgt tgataatcat gacaccgaac ccggccaagc gctgcagtca tgggtcgacc catggttcaa accgttggct tacgccttta ttctaactcg gcaggaagga tacccgtgcg tcttttatgg tgactattat ggcattccac aatataacat tccttcgctg aaaagcaaaa tcgatccgct cctcatcgcg cgcagggatt atgcttacgg aacgcaacat gattatcttg atcactccga catcatcggg tggacaaggg aaggggtcac tgaaaaacca ggatccgggc tggccgcact gatcaccgat gggccgggag gaagcaaatg gatgtacgtt ggcaaacaac acgctggaaa agtgttctat gaccttaccg gcaaccggag tgacaccgtc accatcaaca gtgatggatg gggggaattc aaagtcaatg gcggttcggt ttcggtttgg gttcctagaa aaacgaccgt ttctaccatc gctcggccga tcacaacccg accgtggact ggtgaattcg tccgttggac cgaaccacgg ttggtggcat ggcct SEQ ID NO: 24 (Satori F) 5′-CTCATCTTCTTGCTGCCTCATTCTGCAGCTTC-3′ SEQ ID NO: 25 (Satori R) 5′-TTATCCTTTACCTTGTCTCCAAGC-3′

DETAILED DESCRIPTION I. Introduction

The present composition and methods relate to variants of a parent α-amylase that exhibit an alteration in at least one of the following properties relative to said parent α-amylase: specific activity, substrate specificity, substrate binding, substrate cleavage, thermal stability, pH-dependent activity, pH-dependent stability, oxidative stability, Ca²⁺ dependency, pI, and wash performance. The variants are suitable for starch conversion, ethanol production, laundry washing, dish washing, hard surface cleaning, and other industrial use.

Although numerous mutatutions are described, they have in the common the ability to improve the performance of parental α-amylases that share structural features in terms of amino acid sequence identity and three dimension structure. Several of these mutations have been found to be combinable with other mutations, which make them of particular value in designing variant α-amylases with preselected properties. Also described are positions that are either not amenable to mutation, in general, or not amenable to mutation and combination with other mutions. The identification of these positions is also important in designing variant α-amylases with preselected properties.

Although studies performed in support of the present compositions and methods were performed primarily using a particular parental α-amylase from Geobacillus stearothermophilus, structurally-related α-amylases are likely to benefit from equivalent mutations. Accordingly, the present description provides a roadmap for modifying any of a large number of α-amylases to produce beneficial changes in performance characteristics and for identifying novel α-amylases likely to have desirable performance characteristic based on the presence of certain amino acid residues at specified positions.

These and other aspects and embodiments of the compositions and methods are described in more detail, below.

II. Definitions and Nomenclature

Prior to describing the present compositions and methods in more detail, various terminology, nomenclature, and general principles are set forth, below.

A. Definitions

The following terms and phrases are defined for clarity. Terms and phrases that are not defined should be given their ordinary meaning as used in the art. Reference is made to standard molecular biology references, such as Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL (2nd Ed., 1989); Kreigler, GENE TRANSFER AND EXPRESSION; A LABORATORY MANUAL (1990); Ausubel et al. (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (1994); Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, New York (1994); and Hale and Markham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, N.Y. (1991).

As used herein, the term “starch” refers to any material comprised of the complex polysaccharide carbohydrates of plants, comprised of amylose and amylopectin with the formula (C₆H₁₀O₅)_(x), wherein X can be any number. Exemplary sources of starch include but are not limited to grains, grasses, tubers, and roots, and more specifically wheat, barley, corn, rye, rice, sorghum, brans, cassava, millet, potato, sweet potato, and tapioca.

As used herein, the term “alpha (α)-amylase” refers to enzymes that catalyze the hydrolysis of α-1,4-glucosidic linkages, e.g., E.C. class 3.2.1.1. These enzymes have also been described as effecting the exo or endohydrolysis of 1,4-α-D-glucosidic linkages in polysaccharides containing 1,4-α-linked D-glucose units. Another term used to describe these enzymes is “glycogenases.” Exemplary enzymes include α-1,4-glucan 4-glucanohydrase glucanohydrolase.

As used here, the term “recombinant,” when used in reference to a cell, nucleic acid, protein or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.

As used herein, the terms “protein” and “polypeptide” are used interchangeably to refer to a contiguous chain of amino acid residue linked by peptide bonds. The conventional one-letter or three-letter code for amino acid residues is used.

As used herein, a “signal sequence” refers to a sequence of amino acid residues at the N-termus of a polypeptide, which facilitates the secretion of an extracellular polypeptide outside the cell. The mature form of the extracellular protein lacks the signal sequence, which is cleaved off during the secretion process.

As used herein, a “gene” refers to a DNA segment that is involved in producing a polypeptide and includes regions preceding and following the coding regions as well as intervening sequences (introns) between individual coding segments (exons). The name of a gene is generally italicized, while the name of a corresponding protein is generally not italicized and the first letter is capitalized.

As used herein, the terms “nucleic acid” and “polynucleotide” are used interchangeably to refers to a contiguous chain of nucleosides linked by phosphodiester or similar bonds, and” encompasses DNA, RNA, whether single-stranded, double-stranded, or partially double-stranded, as well as chemically modified DNA or RNA or synthetic derivatives, thereof. Unless other wise specified, the sequences of nucleic acids are present in a 5′ to 3′ direction. The skilled person will appreciate that because the genetic code is degenerate, more than one codon may encode a particular amino acid.

As used herein, a “vector” refers to a polynucleotide sequence designed to introduce nucleic acids into one or more cell types. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, cassettes, and the like.

As used herein, an “expression vector” refers to a DNA construct comprising a DNA sequence that is operably linked to a suitable control sequence capable of effecting expression of the DNA in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome binding sites on the mRNA, enhancers and sequences which control termination of transcription and translation.

As used herein, a “promoter” is a regulatory sequence that is involved in binding RNA polymerase to initiate transcription of a gene. A promoter may be an inducible promoter or a constitutive promoter. An exemplary promoter is from the Trichoderma reesei cbh1 gene, which is an inducible promoter.

As used herein, the term “under transcriptional control” indicates that the transcription of a specified polynucleotide, usually a DNA sequence, depends on its being operably linked to a specified promoter and/or other element(s), which regulate its transcription.

As used herein, the term “under translational control” indicates that the translation of a specified polynucleotide, usually a mRNA sequence, depends on its being operably linked to a specified element(s), which regulate its translation.

As used herein, the term “derived” encompasses the terms “originated from,” “obtained from,” “obtainable from,” and “isolated from,” and is used to indicate that a specified polypeptide, polynucleotide, expression vector, host cell, or the like, is a modified variant of a parental polypeptide, polynucleotide, expression vector, host cell, or the like.

As used herein, “operably-linked” means that the described components are in a relationship permitting them to function in their intended manner. For example, a regulatory sequence can be “operably-linked” to a coding sequence in such a way that expression of the coding sequence is achieved under conditions compatible with the regulatory sequence.

The term “selective/selectable marker” refers to a gene capable of being expressed in a host cell that allows for ease of selection of those host cells using a media component or growth condition. Examples of selectable markers include but are not limited to gene that confer antibiotic/antimicrobial resistance (e.g., hygromycin, bleomycin, or chloramphenicol) and/or genes that confer a metabolic or nutritional advantage.

As used herein, a polynucleotide or a polypeptide has a certain “percent/percentage sequence identity” (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) with another sequence when the specified percentage of bases or amino acid residues are the same following alignment the sequences. Alignment and the percent homology or identity can be determined using any suitable software program known in the art, for example those described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel et al. (eds) (1987) Supplement 30, section 7.7.18). Preferred programs include the Vector NTI Advance™ 9.0 (Invitrogen Corp. Carlsbad, Calif.), GCG Pileup, FASTA (Pearson et al. (1988) Proc. Natl, Acad. Sci. USA 85:2444-2448), and BLAST (BLAST Manual, Altschul et al., Natl Cent. Biotechnol. Inf., Natl Lib. Med. (NCIB NLM NIH), Bethesda, Md., and Altschul et al. (1997) NAR 25:3389-3402) programs. Another preferred alignment program is ALIGN Plus (Scientific and Educational Software, Pa.), preferably using default parameters. Another sequence software program that finds use is the TFASTA Data Searching Program available in the Sequence Software Package Version 6.0 (Genetics Computer Group, University of Wisconsin, Madison, Wis.).

As used herein, a “host strain” or “host cell” refers to an organism suitable for introducing an expression vector or DNA construct comprising a polynucleotide encoding a subject polypeptide. Host cells are preferably bacterial or fungal cells but may also be plant cells (e.g., protoplasts), insect cells, or mammalian cells.

As used herein, the term “culturing” refers to growing a population of microbial cells under suitable conditions in liquid or solid medium. Culturing includes fermentative bioconversion of a starch substrate containing granular starch to an end-product (typically in a vessel or reactor).

As used herein, the term “fermentation” refers to the enzymatic and substantially anaerobic breakdown of organic substances by microorganisms to produce simpler organic compounds. While fermentation generally occurs under anaerobic conditions it is not intended that the term be solely limited to strict anaerobic conditions, as fermentation also occurs in the presence of oxygen.

As used herein, the term “contacting,” with reference to an enzyme and its substrate, refers to the placing of the enzyme in sufficiently close proximity to the substrate to enable the enzyme to convert the substrate to an end-product (i.e., act on the substrate). Contacting can be brought about by mixing solutions or suspensions of enzymes and substrates.

As used herein, the term “enzymatic conversion” generally refers to the modification of a substrate by enzyme action, for example, the modification of a starch substrate by the action of an amylase, glucoamylase, or other enzyme.

As used herein, the term “saccharification” refers to enzymatic conversion of starch to glucose.

As used herein, the term “gelatinization” refers to solubilization of starch (e.g., raw or crystalline starch) by cooking to form a viscous suspension.

As used herein, the term “liquefaction” refers to the stage in starch conversion in which gelatinized starch is hydrolyzed to lower molecular weight soluble dextrins.

As used herein, the term “degree of polymerization (DP)” refers to the number (n) of anhydroglucopyranose units in a given saccharide. Examples of DP1 saccharides are monosaccharides, such as glucose and fructose. Examples of DP2 saccharides are disaccharides, such as maltose and sucrose. A DP>3 saccharide has a degree of polymerization greater than 3.

As used herein, the terms “end-product” or “desired end-product” refer to a molecule that is enzymatically derived from a substrate, such as starch.

As used herein, the term “dry solids content (ds)” refers to the total solids of a slurry expressed in terms of % on a dry weight basis (% wt/wt).

As used herein, the term “slurry” refers to an aqueous mixture containing insoluble solids.

As used herein, the term “residual starch” refers to the remaining starch (soluble or insoluble) in a starch composition after fermentation.

As used herein, a “recycling step” refers to the recycling of mash components, which may include residual starch, enzymes, and/or microorganisms to affect or participate in the fermentation of additional starch compositions.

As used herein, the term “mash” refers to a mixture of fermentable carbon molecules (e.g., carbohydrates) in water, which may be used to produce a fermented product, such as an alcohol. The terms “beer” and “mash” may be used interchangeability.

As used herein, the term “stillage” refers to a mixture of non-fermented solids and water, which is the residue after removal of alcohol from a fermented mash.

As used herein, the terms “distillers dried grain (DDG)” and “distillers dried grain with solubles (DDGS)” refer to a useful by-product of grain fermentation.

As used herein, an “ethanologenic microorganism” refers to a microorganism capable of converting a sugar or oligosaccharide to ethanol. Ethanologenic microorganisms are generally ethanologenic by virtue of their ability to express one or more enzymes that individually or together convert sugar to ethanol.

As used herein, the term “ethanol producer” or ethanol producing microorganism” refers to any organism or cell that is capable of producing ethanol from a hexose or pentose. Generally, ethanol-producing cells contain an alcohol dehydrogenase and a pyruvate decarboxylase. Examples of ethanol producing microorganisms include fungal microorganisms such as yeast. Preferred yeast includes strains of Sacchromyces, particularly, S. cerevisiae.

As used herein, the term “heterologous,” with reference to a polynucleotide or polypeptide, refers to a polynucleotide or polypeptide that does not naturally occur in a host cell. The protein may be a commercially important industrial polypeptide, such as an enzyme. It is intended that the term encompasses polynucleotides and polypeptides that are (or are encoded by) naturally occurring genes, mutated genes, and/or synthetic genes.

As used herein, the term “endogenous,” with reference to a polynucleotide or polypeptide, refers to a polynucleotide or polypeptide that occurs naturally in the host cell.

As used herein, the terms “recovered,” “isolated,” and “separated” refer to a compound, protein, cell, nucleic acid or amino acid that is removed from at least one component with which it is naturally associated.

As used herein, the terms “transformed,” “stably transformed,” and “transgenic,” used in reference to a cell, means the cell includes a non-native (e.g., heterologous) nucleic acid sequence integrated into its genome or carried as an episomal plasmid that is maintained through multiple generations.

As used herein, the term “expression” refers to the process by which a polypeptide is produced based on the nucleotide sequence of a gene. The process includes both transcription and translation.

As used herein, the term “introduced,” in the context of inserting a nucleic acid sequence into a cell, refers to “transfection,” “transformation,” or “transduction,” and includes reference to the incorporation of a nucleic acid sequence into a eukaryotic or prokaryotic cell wherein the nucleic acid sequence may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

As used herein, the term “specific activity” refers to the number of moles of substrate that can be converted to product by an enzyme or enzyme preparation per unit time under specific conditions. Specific activity is generally expressed as units (U)/mg of protein.

As used herein, the term “yield” refers to the amount of end-product produced using a specified method and specified reagents (including enzymes). The amount of end-product may be expressed in terms of mass, volume, concentration, or the like, and may include a reference to the amount of starting material (e.g., substrate), time, or other conditions.

As used herein, there term “performance index (PI)” refers to the ratio of performance of a variant enzyme to the parent or reference enzyme. Within this context, various further terms and phrases are used to characterize the performance of variants: “up mutations” have a PI>1; neutral mutations have a PI>0.5; non-deleterious mutations have a PI>0.05; deleterious mutations have a PI=0.05; combinable mutations have a PI=0.5 for at least one property, and >0.05 for all properties.

As used herein, “combinable mutations” are mutations that can be combined to deliver proteins with preselected performance indices (PI) for one or more desired properties (see above).

As used herein, “ATCC” refers to the American Type Culture Collection located in Manassas, Va., USA.

As used herein, “NRRL” refers to the Agricultural Research Service Culture Collection, National Center for Agricultural Utilization Research (previously known as USDA Northern Regional Research Laboratory) in Peoria, Ill., USA.

In general, numeric ranges are inclusive of the numbers defining the range. The singular articles “a,” “an,” and “the” include plural referents, unless the context clearly dictates otherwise. The headings are provided for ease of reading and should not be construed as confining described subject matter to a portion of the specification. All patents and publications, including all sequences disclosed within such patents and publications, are expressly incorporated by reference.

B. Nomenclature

The conventional one-letter and three-letter codes for amino acid residues are used unless otherwise specified. Variant polypeptides are described using the following nomenclature: original amino acid(s): position(s): substituted amino acid(s).

According to this nomenclature, for instance the substitution of serine by an alanine in position 242 is shown as:

Ser242Ala or S242A

A deletion of alanine in position 30 is shown as:

Ala30* or A30* or ΔA30

And an insertion of an additional amino acid residue, such as lysine, is shown as:

Ala30AlaLys or A30AK

A deletion of a consecutive stretch of amino acid residues, such as amino acid residues 30-33, is indicated as (30-33)* or Δ(A30-N33).

Where a polypeptide contains a “deletion” in comparison with other polypeptides (or a parent polypeptide) and an insertion is made in this position it is indicated as:

-   -   *36Asp or *36D, where the example represents for insertion of an         aspartic acid in position 36.

Multiple mutations are separated by plus signs. For example, mutations at positions 30 and 34, substituting alanine and glutamic acid for asparagine and serine, respectively, are represented by:

Ala30Asp+Glu34Ser or A30N+E34S

When one or more alternative amino acid residues may be inserted in a given position it is indicated as

A30N,E or A30N or A30E

Furthermore, when a position suitable for modification is identified without any specific modification being suggested, it is to be understood that any amino acid residue may be substituted for the amino acid residue present in the position. For instance, when a modification of an alanine in position 30 is mentioned, but not specified, it is to be understood that the alanine may be deleted or substituted for any other amino acid, i.e., any one of:

R, N, D, A, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V.

Further, “A30X” means any one of the following substitutions:

-   -   A30R, A30N, A30D, A30C, A30Q, A30E, A30G, A30H, A301, A30L,         A30K, A30M, A30F, A30P, A30S, A30T, A30W, A30Y, or A30 V; which         may also be presented as:         A30R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V.

If the parent enzyme used for the numbering already has the amino acid residue in question suggested for substitution in that position, the following nomenclature is used:

-   -   “X30N” or “X30N,V” in the case where for instance one or N or V         is present in the wildtype.

Thus, it means that other corresponding parent enzymes are substituted to an “Asn” or “Val” in position 30.

C. Characteristics of Amino Acid Residues

The following general information characteristic of amino acid residues are provided for reference.

Charged Amino Acids:

Asp, Glu, Arg, Lys, H is

Negatively charged amino acids (with the most negative residue first):

Asp, Glu

Positively charged amino acids (with the most positive residue first):

Arg, Lys, H is

Neutral Amino Acids:

Gly, Ala, Val, Leu, lie, Phe, Tyr, Trp, Met, Cys, Asn, Gln, Ser, Thr, Pro

Hydrophobic amino acid residues (with the most hydrophobic residue listed last):

Gly, Ala, Val, Pro, Met, Leu, lie, Tyr, Phe, Trp,

Hydrophilic amino acids (with the most hydrophilic residue listed last):

Thr, Ser, Cys, Gln, Asn

III. α-Amylases for Use in the Present Compositions and Methods

The following paragraphs describe “Spezyme® Xtra-like” or “AmyS-like” α-amylases that can be modified and used according as described herein.

A. Homology Among α-Amylases

Experiments performed in support of the present compositions and methods have been performed using Geobacillus (formerly Bacillus) stearothermophilus α-amylase (i.e., AmyS), exemplified by SEQ ID NO: 2. A variant of this amylase is commercially available as SPEZYME® Xtra (Danisco US Inc, Genencor Division, Palo Alto, Calif., USA).

A number of α-amylases produced by Bacillus spp. are highly homologous (identical) on the amino acid level to AmyS, and the many of mutations described herein are expected to produce similar effects when made in these amylase, which are collectively referred to as “SPEZYME® Xtra-like” α-amylases or “AmyS-like” α-amylases. The identity of a number of known Bacillus α-amylases is summarized in Table A:

TABLE A Percent identity 707 AP1378 BAN BSG SP690 SP722 AA560 LAT 707 100.0 86.4 66.9 66.5 87.6 86.2 95.5 68.1 AP1378 86.4 100.0 67.1 68.1 95.1 86.6 86.0 69.4 BAN 66.9 67.1 100.0 65.6 67.1 68.8 66.9 80.7 BSG 66.5 68.1 65.6 100.0 67.9 67.1 66.3 65.4 SP690 87.6 95.1 67.1 67.9 100.0 87.2 87.0 69.2 SP722 86.2 86.6 68.8 67.1 87.2 100.0 86.8 70.8 AA560 95.5 86.0 66.9 66.3 87.0 86.8 100.0 68.3 LAT 68.1 69.4 80.7 65.4 69.2 70.8 68.3 100.0

The B. licheniformis α-amylase (LAT) having the amino acid sequence shown in SEQ ID NO: 7 has been found to be about 81% homologous with the B. amyloliquefaciens α-amylase having the amino acid sequence shown in SEQ ID NO: 9, and about 65% homologous with the Geobacillus (formerly Bacillus) stearothermophilus α-amylase (BSG; AmyS) comprising the amino acid sequence shown in SEQ ID NO: 1. Further homologous α-amylases include SP690 and SP722 disclosed in WO 95/26397 and the #707 α-amylase derived from Bacillus sp., shown in SEQ ID NO: 6 and described by Tsukamoto et al. (1988) Biochemical and Biophysical Research Communications 151:25-31. The KSM AP1378 α-amylase (SEQ ID NO: 12) is disclosed in WO 97/00324 (from KAO Corporation).

Still further homologous α-amylases include the α-amylase produced by the B. lichenformis strain described in EP 0 252 666. (ATCC 27811), and the α-amylases identified in WO 91/00353 and WO 94/18314. Other commercial SPEZYME® Xtra-like α-amylases are comprised in the products sold under the following tradenames: SPEZYME® AA and Ultraphlow (available from Danisco US Inc, Genencor Division), and KEISTASE™ (available from Daiwa) and LIQUEZYME® SC (SEQ ID NO: 15) available from Novozymes, DK). Other related α-amylases include Termamyl® (SEQ ID NO: 8; Novozymes), STAINZYME™ (SEQ ID NO: 10; Novozymes), NATALASE™ (SEQ ID NO: 11; Novozymes), KAO KSM K38 (SEQ ID NO: 13), KAO KSM K36 (SEQ ID NO: 14), other α-amylases mentioned in the Table, and other α-amylases described herein.

Because of the substantial homology found between these α-amylases, they are considered to belong to the same class of α-amylases and are encompassed by the present compositions and methods. While G. stearothermophilus α-amylase (SEQ ID NO: 2) is used as a starting point, corresponding positions in these and other α-amylases, e.g., the SP722, BLA, BAN, AA560, SP690, KSM AP1378, #707 and other Bacillus α-amylases are also expected to benefit from modifications to be described.

Accordingly, the terms SPEZYME® Xtra-like α-amylase or AmyS-like α-amylase are intended to include an α-amylase having the amino acid sequence of SEQ ID NOs: 1, 2, 6, 7, 8, 9, 10, 11, 12, 15, and 16. In some embodiments, SPEZYME® Xtra-like α-amylase or AmyS-like α-amylase also include α-amylases that exhibits substantial identity at the amino acid level to SEQ ID NO: 2, for example, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99% homology (identity). In further embodiments, SPEZYME® Xtra-like α-amylase or AmyS-like α-amylase also include α-amylases that exhibits substantial identity at the amino acid level to one or more of SEQ ID NOs: 1, 2, 6, 7, 8, 9, 10, 11, 12, 15, and 16, for example, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99% homology (identity).

The homology of known or suspected α-amylases to a reference α-amylase may be determined by means of computer programs known in the art. Generally, a structural alignment between SPEZYME® Xtra (SEQ ID NO: 2) and, e.g., another α-amylase may be used to identify equivalent/corresponding positions in other SPEZYME® Xtra-like α-amylases, which can be mutated as described herein to produce similar effects. One exemplary program is GAP, which is provided in the GCG program package (described above). In particular, Gap GCG v8 may be used with the default scoring matrix for identity and the following default parameters: GAP creation penalty of 5.0 and GAP extension penalty of 0.3, respectively for nucleic acidic sequence comparison, and GAP creation penalty of 3.0 and GAP extension penalty of 0.1, respectively, for protein sequence comparison. GAP uses the method of Needleman and Wunsch, (1970), J. Mol. Biol. 48:443-453, to make alignments and to calculate the identity. Other programs and methods are known in the art.

Another method of obtaining structural alignments is to use the Pile Up programme from the GCG package using default values of gap penalties, i.e., a gap creation penalty of 3.0 and gap extension penalty of 0.1. Other structural alignment methods include the hydrophobic cluster analysis (Gaboriaud et al., (1987), FEBS LETTERS 224, pp. 149-155) and reverse threading (Huber, T; Torda, A E, PROTEIN SCIENCE Vol. 7, No. 1 pp. 142-149 (1998).

SPEZYME® Xtra-like α-amylases or AmyS-like α-amylases further include polypeptides encoded by a DNA sequence that hybridizes to a DNA sequence encoding one or more of the aforementioned α-amylases, as exemplified by SEQ ID NOs: 9 (BAN), (BSG; AmyS), 3 (SP722), 1 (SP690), 7 (LAT), and 11 (AA560) of WO 06/002643 and polynucleotides encoding the amino acid sequences of SEQ ID NOs: 1, 2, 6, 7, 8, 9, 10, 11, 12, 15 and 16. A nucleic acid is hybridizable to another nucleic acid sequence when a single stranded form of the nucleic acid can anneal to the other nucleic acid under appropriate conditions of temperature and solution ionic strength. Hybridization and washing conditions are well known in the art (see, e.g., Sambrook (1989) supra, particularly chapters 9 and 11). An oligonucleotide probe used in the characterization of a known or suspected SPEZYME® Xtra-like α-amylase above may suitably be prepared on the basis of the full or partial nucleotide or amino acid sequence of the α-amylase in question.

Suitable conditions for testing hybridization involve pre-soaking in 5×SSC and prehybridizing for 1 hour at 40° C. in a solution of 20% formamide, 5×Denhardt's solution, 50 mM sodium phosphate, pH 6.8, and 50 mg of denatured sonicated calf thymus DNA, followed by hybridization in the same solution supplemented with 100 mM ATP for 18 hours at 40° C., followed by three times washing of the filter in 2×SSC, 0.2% SDS at 40° C. for 30 minutes (low stringency), preferred at 50° C. (medium stringency), more preferably at 65° C. (high stringency), even more preferably at 75° C. (very high stringency). More details about the hybridization method can be found in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989. In some embodiments, stringent conditions correspond to a Tm of 65° C. and 0.1×SSC, 0.1% SDS.

B. Parental α-Amylases

Any of the aforementioned SPEZYME® Xtra/AmyS-like α-amylases may serve as a parental α-amylase to be modified and used as described herein. The parental/parent α-amylase may also be referred to as a “backbone” or “template,” and variant amylases may be derived, therefrom. In some embodiments, the parent α-amylase is derived from G. stearothermophilus. In a particular embodiment, the parent α-amylase has the amino acid sequence of SEQ ID NO: 2. In other embodiments, the parental α-amylase has the amino acid sequence of SEQ ID NOs: 1, 6, 7, 8, 9, 10, 11, 12, 15, and 16, or exhibits substantial identity at the amino acid level to SEQ ID NOs: 1, 2, 6, 7, 8, 9, 10, 11, 12, 15, and/or 16, for example, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99% homology (identity).

A parental α-amylase may also be a hybrid α-amylase, i.e., an α-amylase, which comprises a combination of partial amino acid sequences derived from at least two α-amylases, such as those described, above. In addition the hybrid α-amylase may include a portion of a SPEZYME® Xtra/AmyS-like α-amylase and portion of one or more other α-amylases of microbial (bacterial or fungal) and/or mammalian origin.

Thus, the parent hybrid α-amylase may comprise a combination of partial amino acid sequences deriving from at least two SPEZYME® Xtra-like α-amylases, or from at least one SPEZYME® Xtra-like and at least one non-SPEZYME® Xtra-like bacterial α-amylase, or from at least one SPEZYME® Xtra-like and at least one fungal α-amylase, and so forth. For instance, the parent α-amylase may comprise a C-terminal part of an α-amylase derived from a strain of B. licheniformis, and an N-terminal part of an α-amylase derived from a strain of G. stearothermophilus.

IV. Altered Properties of α-Amylase Variants

The following section describes the relationship between mutations, which are present in the variant polypeptides described herein, and desirable alterations in properties (relative to those of a parent SPEZYME® Xtra-like α-amylase), which result therefrom.

In a first aspect a variant of a parent G. stearothermophilus α-amylase is provided, comprising an alteration at one or more positions (using SEQ ID NO: 1 or SEQ ID NO: 2 for the amino acid numbering) selected from the group of:

-   -   P17, D19, T21, N28, S51, G72, V74, A82, Q86, Q89, A93, G95, Q97,         W115, D117, P123, S124, D125, N127, I130, G132, Q135, P145,         G146, G148, S153, Y159, W166, S169, K171, W187, P209, N224,         S242, G256, D269, N271, T278, N281, G302, A304, R308, T321,         Q358, P378, S382, K383, T398, H405, T417, E418, P420, G421,         P432, W437, G446, G454, S457, T459, T461, S464, G474, R483.         wherein

(a) the alteration(s) are independently

-   -   (i) an insertion of an amino acid downstream of the amino acid         which occupies the position,     -   (ii) a deletion of the amino acid which occupies the position,         or     -   (iii) a substitution of the amino acid which occupies the         position with a different amino acid,

(b) the variant has α-amylase activity and (c) each position corresponds to a position of the amino add sequence of the parent G. stearothermophilus α-amylase having the amino acid sequence shown in SEQ ID NO: 1 or 2.

Specifically contemplated herein are S242A, S242Q, S242N and S242E, which may be combined with mutations at R179, G180, 1181, G182, and/or K183, associated with calcium-sodium binding, and/or a mutations at P245 in the middle of an α-helix.

Corresponding positions in other parent SPEZYME® Xtra-like α-amylases can be found by alignment as described above and shown in the alignment in FIG. 4.

Stability

In the context of the variants described herein, mutations (including amino acid substitutions and deletion) of importance with respect to achieving altered stability, in particular improved stability (i.e., higher or lower), at especially high temperatures (i.e., 70-120° C.) and/or extreme pH (i.e., low or high pH, i.e, pH 4-6 or pH 8-11, respectively), in particular at free (i.e., unbound, therefore in solution) calcium concentrations below 60 ppm, include any of the mutations listed in the “Altered Properties” section. The stability may be determined as described in the “Methods” section below.

Exemplary mutations in SPEZYME® Extra and related α-amylases that increase stability include:

(a) a substitution that introduces one or more of the amino acid residues: 74A, 115L, 124K, 124R, 132A, 132C, 135A, 145A, 146A, 148A, 148N, 159A, 159C, 159D, 159E, 159F, 159G, 159H, 159K, 159L, 159N, 159R, 159S, 159T, 159V, 169A, 169L, 169M, 169Y, 179A, 181A, 181C, 181D, 181E, 181L, 181P, 181Q, 181V, 181Y, 242A, 242D, 242E, 242Q, 261L, 271A, 271V, 278A, 278H, 278K, 278N, 278R, 281A, 281L, 281M, 302D, 302M, 304D, 304E, 304M, 321A, 321H, 321Q, 321R, 333Q, 378D, 378N, 378R, 382D, 398A, 418A, 418M, 418N, 420A, 421R, 432A, 432D, 432L, 432M, 432N, 432Q, 432R, 432Y, 437D, 437G, 437H, 437L, 437M, 437Y, 446A, 446Y, 454A, 464Q, 464Y, 474A, 474E, 474K, 474L, 474M, 474N, 474P, 474Q, 474R, 474S, and 474V, or

(b) a substitution that introduces one or more of the amino acid residues: 6I, 6N, 6Q, 6T, 6V, 14T, 16F, 25A, 25C, 25G, 25Q, 27M, 36Q, 36S, 39G, 39V, 50I, 50L, 50M, 50N, 50Q, 52S, 53T, 67N, 67S, 80D, 80I, 90E, 133P, 133V, 137M, 137S, 141E, 141I, 141L, 141M, 141Q, 141R, 141S, 141V, 150E, 1511, 152G, 155S, 155Y, 168W, 173T, 188P, 193F, 193K, 193L, 193Y, 213L, 213M, 213V, 217Q, 220P, 220Q, 220R, 220S, 220V, 221I, 221S, 249E, 250F, 250I, 250M, 252L, 253Y, 254E, 254F, 254T, 254V, 255F, 255K, 255W, 257L, 257M, 257S, 257V, 258D, 258G, 258H, 258K, 258Q, 258T, 258V, 268F, 274W, 283M, 283N, 283V, 285E, 285Q, 293G, 293K, 294W, 301F, 301I, 301P, 301R, 301T, 301W, 309D, 309V, 312H, 312S, 312V, 312Y, 313G, 313H, 313I, 313L, 313S, 313V, 318T, 338A, 338C, 338G, 338M, 338T, 339K, 339T, 339V, 340A, 340M, 340Q, 340T, 343C, 343I, 343P, 343R, 343Y, 345I, 345Q, 369I, 369T, 370G, 375T, 385T, 386K, 394L, 394V, 400A, 400N, 400V, 402H, 4021, 402T, 402V, 402W, 403A, 403E, 403G, 403Q, 403R, 403T, 403V, 404C, 404E, 404G, 404I, 404V, 419A, 419C, 419M, 419T, 422E, 422G, 433A, 433H, 4331, 433K, 433L, 433M, 433V, 433Y, 442A, 442G, 442N, 442R, 442S, 442T, 442V, 442W, 442Y, 445G, 4451, 445N, 445T, 445V, 445W, 447I, 447N, 447Q, 447W, 447Y, 448C, 448F, 448G, 448H, 4481, 448N, 448Y, 450C, 450H, 450M, 450N, 450R, 450S, 450T, 450W, 455G, 455I, 455P, 455V, 463A, 463M, 463S, 463T, 463V, 463W, 465G, 465I, 465K, 465N, 465T, 465V, 469D, 469W, 469Y, 471I, 471V, 473G, 473Y, 476A, 476G, 476L, 476M, 476N, and 476T

Ca²⁺ Stability

Altered Ca²⁺ stability means the stability of the enzyme under Ca²⁺ depletion has been improved, i.e., higher or lower stability. In the context of the presently described variants, mutations (including amino acid substitutions and deletions) of importance with respect to achieving altered Ca²⁺ stability, in particular improved Ca²⁺ stability, i.e., higher or lower stability, at especially high pH (i.e., pH 8-10.5) include any of the mutations listed in the in “Altered Properties” section.

Specific Activity and or Increased Expression

In a further aspect, important mutations (including amino acid substitutions and deletions) with respect to obtaining variants exhibiting altered specific activity, in particular increased or decreased specific activity, especially at temperatures from 10-60° C., preferably 20-50° C., especially 30-40° C., include any of the mutations listed in the in “Altered properties” section. The specific activity may be determined as described in the “Methods” section below. In some cases, the mutations increase expression rather than or in addition to increasing specific activity. Exemplary mutations are as follows:

(a) a substitution that introduces one or more of the amino acid residues selected from the group consisting of 124N, 125A, 125K, 125N, 130A, 130S, 159A, 159D, 159E, 159G, 159H, 159K, 159L, 159N, 159R, 159S, 159T, 166F, 166G, 166H, 166S, 166Y, 169L, 179A, 179P, 180A, 180D, 180H, 180K, 180L, 180N, 180T, 180V, 180Y, 181A, 181D, 181E, 181G, 181P, 181R, 181S, 181V, 187A, 187C, 187K, 187N, 187P, 187Q, 187R, 187S, 242H, 242N, 278H, 278K, 278N, 278R, 281M, 302D, 304M, 304Y, 321H, 321Q, 321R, 333Q, 432Q, 437Y, 446A, 474Q, and 474S, or

(b) a substitution that introduces one or more of the amino acid residues selected from the group consisting of 6A, 6D, 6E, 6H, 6I, 6K, 6L, 6M, 6N, 6P, 6Q, 6R, 6S, 6T, 6V, 6W, 6Y, 13K, 14F, 14T, 14Y, 15A, 15D, 15E, 015G, 15H, 15K, 15N, 15P, 15Q, 15R, 15S, 15T, 15W, 16A, 16E, 16G, 16H, 16K, 16N, 16P, 16Q, 16R, 16T, 25C, 39D, 39E, 39N, 39Q, 81Y, 121P, 139D, 139H, 139R, 139Y, 177A, 188D, 191H, 191K, 192A, 192D, 192G, 192N, 192P, 192Q, 192S, 192T, 192V, 192Y, 196A, 196C, 196D, 196E, 196F, 196H, 1961, 196K, 196P, 196R, 196S, 196T, 196V, 201A, 201E, 201G, 201H, 201M, 202H, 216E, 216G, 216H, 216M, 216Q, 216R, 216S, 216T, 216Y, 221A, 221D, 221F, 221I, 221L, 221M, 221N, 221R, 221S, 221V, 221Y, 237G, 240G, 240N, 240P, 240Q, 240R, 240T, 246R, 250A, 250D, 250E, 250F, 250G, 250I, 250K, 250L, 250M, 250N, 250Q, 250R, 250S, 250W, 252K, 268A, 268D, 268E, 268G, 268H, 268K, 268N, 268P, 268Q, 268R, 268S, 274A, 274D, 274G, 274I, 274K, 274L, 274N, 274Q, 274R, 274S, 274T, 275K, 285Q, 285Y, 293K, 293R, 318A, 318F, 318G, 3181, 318K, 318L, 318M, 318R, 318S, 318T, 318V, 318Y, 319C, 319D, 319H, 319I, 319K, 319R, 319Y, 320K, 320R, 320T, 338A, 338G, 338I, 338M, 338P, 338S, 338V, 339G, 339P, 340A, 340D, 340E, 340H, 340K, 340N, 340Q, 345E, 363D, 363E, 363M, 363N, 363Q, 363S, 366Q, 370A, 370D, 370E, 370H, 370K, 370N, 370Q, 370S, 375A, 375D, 375E, 375K, 375N, 375Q, 375R, 375S, 419A, 419I, 419M, 419P, 419S, 419V, 448Y, 452N, 452Q, 452R, 452S, 471R, and 471Y.

Oxidation Stability

The described variants may have altered oxidation stability, in particular higher oxidation stability, in comparison to the parent α-amylase. Increased oxidation stability is advantageous in, e.g., detergent compositions and decreased oxidation stability may be advantageous in composition for starch liquefaction. Oxidation stability may be determined as described in the “Methods” section below.

Altered pH Profile

Important positions and mutations with respect to obtaining variants with altered pH profile, in particular improved activity at especially high pH (i.e., pH 8-10.5) or low pH (i.e., pH 4-6) include mutations of amino residues located close to the active site residues. In some cases, the improved activity is observed, e.g., at pH<6, at pH<5, or at pH>9.

Preferred specific mutations/substitutions are the ones listed above in the section “Altered Properties” for the positions in question. Suitable assays are described in the “Methods” section below.

Wash Performance

Important positions and mutations with respect to obtaining variants with improved wash performance at especially high pH (i.e., pH 8.5-11) include the specific mutations/substitutions listed above in the section “Altered Properties” for the positions in question. The wash performance may be tested as described below in the “Methods” section.

Starch Liquifaction

Some mutations have the effect of reducing the viscosity of a starch composition compared that observed using a “wild-type” α-amylase, such as SPEZYME® Extra. Exemplary mutations include a substitution that introduces one or more of the amino acid residues selected from the group consisting of I181A, I181P, I181C, I181E, I181Y, S242A, S242E, S242Q, G132A, N193Y, and E188P.

Other Mutations in Variants

In some embodiments, the present variants include one or more modifications in addition to those outlined above. For example, it may be advantageous that one or more proline residues is replaced with a non-proline residue. Exemplary non-proline residues include alanine, glycine, serine, threonine, valine, and leucine. Similarly, it may be advantageous to replace one or more cysteine residues with a non-cysteine residue. Exemplary non-cysteine residues include serine, alanine, threonine, glycine, valine, and leucine.

Furthermore, it may be advantageous to introduce mutations at one or more of the following positions (using SEQ ID NO: 7 for the numbering): M15, V128, A111, H133, W138, T149, M197, N188, A209, A210, H405, T412, in particular the following single, double or triple or multi mutations:

M15X, in particular M15T,L;

V128X, in particular V128E;

H133X, in particular H133Y;

N188X, in particular N188S,T,P;

M197X, in particular M197T,L;

A209X, in particular A209V;

M197T/W138F; M197T/138Y; M15T/H133Y/N188S;

M15N128E/H133Y/N188S; E119C/S130C; D124C/R127C; H133Y/T1491;

G475R, H133Y/S187D; H133Y/A209V.

In the case of the parent α-amylase having the amino acid sequence shown in SEQ ID NO: 7, relevant amino acid residues which may be deleted or substituted with a view to improving the oxidation stability include the single cysteine residue (C363) and the methionine residues located in positions M8, M9, M96, M200, M206, M284, M307, M311, M316 and M438 in SEQ ID NO: 2.

With respect to increasing the thermal stability of an α-amylase variant relative to its parent α-amylase, it appears to be particularly desirable to delete at least one, and preferably two or even three, of the following amino acid residues in the amino acid sequence shown in SEQ ID NO: 2 are F178, R179, G180, I181, G182 and K183. Particularly valuable pairwise deletions of this type are R179*+ G180*; and 1181*+G182* (SEQ ID NO. 16 or 15, respectively) (or equivalents of these pairwise deletions in another α-amylase meeting the requirements of a parent α-amylase in the context of the present disclosure).

Other mutations of interest include N193F and V416G, as exemplified in the amino acid sequence shown as SEQ ID NO: 2.

V. Methods of Preparing α-Amylase Variants

Several methods for introducing mutations into genes are known in the art. After a brief discussion of the cloning of α-amylase-encoding DNA sequences, methods for generating mutations at specific sites within the α-amylase-encoding sequence will be discussed.

A. Cloning and Expression of Nucleic Acids Encoding an α-Amylase

The DNA sequence encoding a parent α-amylase may be isolated from any cell or microorganism producing the α-amylase in question, using various methods well known in the art. First, a genomic DNA and/or cDNA library should be constructed using chromosomal DNA or messenger RNA from the organism that produces the α-amylase to be studied. Then, if the amino acid sequence of the α-amylase is known, homologous, labelled oligonucleotide probes may be synthesized and used to identify α-amylase-encoding clones from a genomic library prepared from the organism in question. Alternatively, a labelled oligonucleotide probe containing sequences homologous to a known α-amylase gene could be used as a probe to identify α-amylase-encoding clones, using hybridization and washing conditions of lower stringency.

Another method for identifying α-amylase-encoding clones would involve inserting fragments of genomic DNA into an expression vector, such as a plasmid, transforming α-amylase-negative bacteria with the resulting genomic DNA library, and then plating the transformed bacteria onto agar containing a substrate for α-amylase, thereby allowing clones expressing the α-amylase to be identified.

Alternatively, the DNA sequence encoding the enzyme may be prepared synthetically by established standard methods, e.g. the phosphoamidite method described by S. L. Beaucage and M. H. Caruthers (1981) or the method described by Matthes et al. (1984). In the phosphoamidite method, oligonucleotides are synthesized, e.g. in an automatic DNA synthesizer, purified, annealed, ligated and cloned in appropriate vectors.

Finally, the DNA sequence may be of mixed genomic and synthetic origin, mixed synthetic and cDNA origin or mixed genomic and cDNA origin, prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate, the fragments corresponding to various parts of the entire DNA sequence), in accordance with standard techniques. The DNA sequence may also be prepared by polymerase chain reaction (PCR) using specific primers, for instance as described in U.S. Pat. No. 4,683,202.

B. Site-Directed Mutagenesis

Once an α-amylase-encoding DNA sequence has been isolated, and desirable sites for mutation identified, mutations may be introduced using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation sites; mutant nucleotides are inserted during oligonucleotide synthesis. In a specific method, a single-stranded gap of DNA, bridging the α-amylase-encoding sequence, is created in a vector carrying the α-amylase gene. Then the synthetic nucleotide, bearing the desired mutation, is annealed to a homologous portion of the single-stranded DNA. The remaining gap is then filled in with DNA polymerase I (Klenow fragment) and the construct is ligated using T4 ligase.

Another method of introducing mutations into α-amylase-encoding DNA sequences involves the 3-step generation of a PCR fragment containing the desired mutation introduced by using a chemically synthesized DNA strand as one of the primers in the PCR reactions. From the PCR-generated fragment, a DNA fragment carrying the mutation may be isolated by cleavage with restriction endonucleases and reinserted into an expression plasmid.

Alternative methods for providing variants include gene shuffling, e.g., as described in WO 95/22625 (from Affymax Technologies N.V.) or in WO 96/00343 (from Novo Nordisk A/S), or other corresponding techniques resulting in a hybrid enzyme comprising the mutation(s), e.g., substitution(s) and/or deletion(s), in question.

C. Expression of α-Amylase Variants

A DNA sequence encoding a variant produced by methods described above, or by alternative methods, can be expressed using an expression vector, which typically includes control sequences encoding a promoter, operator, ribosome binding site, translation initiation signal, and, optionally, a repressor gene or various activator genes.

The recombinant expression vector carrying the DNA sequence encoding an α-amylase variant may be any vector, which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, a bacteriophage or an extrachromosomal element, minichromosome or an artificial chromosome. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.

The DNA sequence should be operably connected to a suitable promoter sequence. The promoter may be any DNA sequence, which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Examples of suitable promoters for directing the transcription of the DNA sequence encoding an α-amylase variant, especially in a bacterial host, are the promoter of the lac operon of E. coli, the Streptomyces coelicolor agarase gene dagA promoters, the promoters of the Bacillus licheniformis α-amylase gene (amyL), the promoters of the G. stearothermophilus maltogenic amylase gene (amyM), the promoters of the B. amyloliquefaciens α-amylase (amyQ), the promoters of the B. subtilis xylA and xylB genes etc. For transcription in a fungal host, examples of useful promoters are those derived from the gene encoding A. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. niger neutral α-amylase, A. niger acid stable α-amylase, A. niger glucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase or A. nidulans acetamidase.

The expression vector may also comprise a suitable transcription terminator and, in eukaryotes, polyadenylation sequences operably connected to the DNA sequence encoding an α-amylase variant. Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter.

The vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUB110, pE194, pAMB1 and pIJ702.

The vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, such as the dal genes from B. subtilis or B. licheniformis, or one which confers antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracyclin resistance. Furthermore, the vector may comprise Aspergillus selection markers such as amdS, argB, niaD and sC, a marker giving rise to hygromycin resistance, or the selection may be accomplished by co-transformation, e.g., as described in WO 91/17243.

While intracellular expression may be advantageous in some respects, e.g., when using certain bacteria as host cells, it is generally preferred that the expression is extracellular. In general, the Bacillus α-amylases mentioned herein comprise a preregion permitting secretion of the expressed protease into the culture medium. If desirable, this preregion may be replaced by a different preregion or signal sequence, conveniently accomplished by substitution of the DNA sequences encoding the respective preregions.

The procedures used to ligate the DNA construct encoding an α-amylase variant, the promoter, terminator and other elements, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989).

The cell comprising a DNA construct or an expression vector is advantageously used as a host cell in the recombinant production of an α-amylase variant. The cell may be transformed with the DNA construct encoding the variant, conveniently by integrating the DNA construct (in one or more copies) in the host chromosome. This integration is generally considered to be an advantage as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g., by homologous or heterologous recombination. Alternatively, the cell may be transformed with an expression vector as described above in connection with the different types of host cells.

The cell may be a cell of a higher organism such as a mammal or an insect, but is preferably a microbial cell, e.g., a bacterial or a fungal (including yeast) cell.

Examples of suitable bacteria are Gram-positive bacteria such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Geobacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus megaterium, Bacillus thuringiensis, or Streptomyces lividans or Streptomyces murinus, or gram-negative bacteria such as E. coli. The transformation of the bacteria may, for instance, be effected by protoplast transformation or by using competent cells in a manner known per se.

The yeast organism may favorably be selected from a species of Saccharomyces or Schizosaccharomyces, e.g. Saccharomyces cerevisiae. The filamentous fungus may advantageously belong to a species of Aspergillus, e.g., A. oryzae or A. niger. Fungal cells may be transformed by a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in a manner known per se. A suitable procedure for transformation of Aspergillus host cells is described in EP 238 023.

An aspect of the present compositions and methods relates producing an α-amylase variant by cultivating a host cell under conditions conducive to the production of the variant amylase and recovering the variant amylase from the cells and/or culture medium. The medium used to cultivate the cells may be any conventional medium suitable for growing the host cell in question and obtaining expression of the α-amylase variant. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g., as described in catalogues of the ATCC).

The α-amylase variant secreted from the host cells may conveniently be recovered from the culture medium by well-known procedures, including separating the cells from the medium by centrifugation or filtration, and precipitating proteinaceous components of the medium by means of a salt such as ammonium sulphate, followed by the use of chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.

VI. Industrial Applications

The present variant α-amylases possess valuable properties allowing for a variety of industrial applications. For example, the variants may be used for starch processing/conversion, e.g., for starch liquefaction (see, e.g., U.S. Pat. No. 3,912,590, EP patent application nos. 252 730 and 63 909, WO 99/19467, and WO 96/28567 all references hereby incorporated by reference). The variants may further be useful in the production of sweeteners and ethanol (see, e.g., U.S. Pat. No. 5,231,017 hereby incorporated by reference), such as fuel, drinking and industrial ethanol, from starch or whole grains. The variants may further be useful for beer making or brewing. The variants may be in the form of compositions, which may further include, e.g., a glucoamylase, a pullulanase, and another α-amylase, in addition to suitable buffers, stabilizing agents, preservatives, and the like.

The amylase variants are also useful in laundry and dishwashing and hard surface cleaning, as components of detergent compositions. The variants may also be useful for desizing of textiles, fabrics and garments (see, e.g., WO 95/21247, U.S. Pat. No. 4,643,736, EP 119,920 hereby incorporated by reference), and in pulp and paper production.

These and other uses of the present compositions and methods are described in more detail, below.

A. Grain Processing and Starch Conversion Applications

Grain processing and starch conversion applications are divided into two categories, namely (1) general starch conversion, which covers the conversion of starch into, e.g., maltodextrins, dextrose syrup, and high fructose syrup, (2) ethanol production, and (3) bearmaking. Although many steps involved in these processes are the similar, they are described separately. In some cases, the variant α-amylase is used in combination with a phytase (4). Compositions for performing these applications are also described (5).

1. General Starch Conversion

Conventional starch-conversion processes, such as liquefaction and saccharification processes are described, e.g., in U.S. Pat. No. 3,912,590 and EP patent publications Nos. 252,730 and 63,909, hereby incorporated by reference. Generally, the starch conversion process degrades starch to lower molecular weight carbohydrate components. In the case of converting starch into a sugar, the starch is depolymerized in a process involving of a pre-treatment step and two or three consecutive process steps, i.e., a liquefaction process, a saccharification process, and depending on the desired end product, and optional isomerization process.

a. Pre-Treatment of Native/Raw Starch

Native/raw starch consists of microscopic granules, which are insoluble in water at room temperature. When an aqueous starch slurry is heated, the granules swell and eventually burst, dispersing the starch molecules into the solution. During this “gelatinization” process, there is a dramatic increase in viscosity. As the solids level is 30-40% in a typical industrial process, the starch has to be thinned or “liquefied” so that it can be handled. This reduction in viscosity is conventionally obtained by enzymatic degradation.

b. Liquefaction

During the liquefaction step, long-chained starch molecules are degraded into shorter branched and linear molecules (maltodextrins) by an α-amylase. The liquefaction process is generally carried out at about 105-110° C. for about 5 to 10 minutes followed by 1-2 hours at 95° C. The pH is typically between about 5.5 and 6.2. To ensure optimal enzyme stability under these conditions, 1 mM of calcium is typically added (40 ppm free calcium ions). After this treatment the liquefied starch will have a “dextrose equivalent” (DE) of 10-15.

c. Saccharification

After the liquefaction process, the maltodextrins are converted into dextrose by addition of a glucoamylase (e.g., OPTIDEX® L-400) and a debranching enzyme, such as an isoamylase (U.S. Pat. No. 4,335,208) or a pullulanase. Before this step the pH is reduced to a value below about 4.5, while maintaining the high temperature (above 95° C.), to inactivate the liquefying α-amylase to reduce the formation of short oligosaccharides called “panose precursors” which cannot be hydrolyzed properly by the debranching enzyme. The temperature is lowered to 60° C., and glucoamylase and debranching enzyme are added. The saccharification process proceeds for 24-72 hours.

Normally, when denaturing the α-amylase after the liquefaction step, about 0.2-0.5% of the saccharification product is the branched trisaccharide Glc pα1-6Glc pα1-4Glc (panose) which cannot be degraded by a pullulanase. If active amylase from the liquefaction step is present during saccharification (i.e., no denaturing), this level can be as high as 1-2%, which is highly undesirable as it lowers the saccharification yield significantly.

d. Isomerization

When the desired final sugar product is, e.g., high fructose syrup the dextrose syrup may be converted into fructose. After the saccharification process, the pH is increased to a value in the range of about 6-8, preferably pH 7.5, and the calcium is removed by ion exchange. The dextrose syrup is then converted into high fructose syrup using, e.g., an immmobilized glucose isomerase (such as GENSWEET® IGI-HF).

2. Ethanol Production

In general alcohol production (ethanol) from whole grain can be separated into 4 main steps, i.e, (a) milling, (b) liquefaction, (c) saccharification, and (d) fermentation. Some of these steps are similar to those described, above.

a. Milling and Slurry Production

A starch-containing substrate, such as grain, corn, milo, or the like, is milled in order to open up the structure and allow for further processing. The two processes used are generally referred to as wet or dry milling. In dry milling, the whole kernel is milled and used in the remaining part of the process. Wet milling gives a very good separation of germ and meal (starch granules and protein) and is with a few exceptions applied at locations where there is a parallel production of syrups.

The milled starch-containing material is be combined with water and recycled thin-stillage resulting in an aqueous slurry. The slurry will comprise between 15 to 55% ds w/w (e.g., 20 to 50%, 25 to 50%, 25 to 45%, 25 to 40%, 20 to 35% and 30-36% ds). The recycled thin-stillage (backset) is typically in the range of 10 to 70% v/v (e.g., 10 to 60%, 10 to 50%, 10 to 40%, 10 to 30%, 10 to 20%, 20 to 60%, 20 to 50%, 20 to 40% and also 20 to 30%). 25 to 40% ds is fairly common.

Once the milled starch-containing material is combined with water and backset, the pH is generally not adjusted in the slurry. Further, the pH is not adjusted after the addition of phytase (see below) and α amylase to the slurry. The pH of the slurry will typically be in the range of pH 4.5 to less than 6.0 (e.g., pH 4.5 to 5.8, pH 4.5 to 5.6, pH 4.8 to 5.8, pH 5.0 to 5.8, pH 5.0 to 5.4, pH 5.2 to 5.5 and pH 5.2 to 5.9). The pH of the slurry may be between pH 4.5 and 5.2 depending on the amount of thin stillage added to the slurry and the type of material comprising the thin stillage. For example, the pH of the thin stillage may be between pH 3.8 and pH 4.5. Table B illustrates the pH change that occurs with addition of increasing amounts of thin stillage to a whole ground corn slurry (32% ds) after stirring for 2 hours at 155° F.

TABLE B pH change that occurs with addition of increasing amounts of thin stillage Thin stillage w/w % Final pH 0 5.52 20 5.29 40 5.16 50 5.09 60 5.05 80 4.98 100 4.94

It should be mentioned that during ethanol production, acids can be added to lower the pH in the beer well to reduce the risk of microbial contamination prior to distillation.

In some cases, phytase is added to the slurry. Phytases are described in more detail, below. In some cases, an α-amylase is added to the slurry. In some cases, a phytase and an α amylase are added to the slurry sequentially. In some cases, a phytase and an α-amylase are added simultaneously. In some cases, the slurry comprising the phytase and α-amylase are incubated (pretreated) for a period of 5 minutes to 8 hours (e.g., 5 minutes to 6 hours, 5 minutes to 4 hours 5 minutes to 2 hours, and 15 minutes to 4 hours). In other cases the slurry are incubated at a temperature in the range of 40 to 115° C., (e.g. 45 to 80° C., 50 to 70° C., 50 to 75° C., 60 to 110° C., 60 to 95° C., 70 to 110° C., 70 to 85° C. and 77 to 86° C.).

In some cases, the slurry is incubated at a temperature of 0 to 30° C. (e.g., 0 to 25° C., 0 to 20° C., 0 to 15° C., 0 to 10° C. and 0 to 5° C.) below the starch gelatinization temperature of the starch-containing material. In some cases, the temperature is below 68° C., below 65° C., below 62° C., below 60° C., or even below 55° C. In some embodiments, the temperature is above 45° C., above 50° C., above 55° C., and even above 60° C. Incubation of the slurry comprising a phytase and an α-amylase at a temperature below the starch gelatinization temperature may be referred to as a primary (1°) liquefaction.

Currently, it is believed that commercially available microbial α-amylases used in the liquefaction process are not sufficiently stable to produce liquefied starch substrate from a dry mill process using whole ground grain at a temperature above about 80° C. at a pH less than pH 5.6. Generally, the stability of many commercially available α-amylases is reduced at a pH of less than about 4.0.

b. Liquefaction

In the liquefaction process, the starch granules are solubilized by hydrolysis to maltodextrins mostly of a DP higher than 4. The raw material can be milled whole grain or a side stream from starch processing. Milled and liquefied grain is also known as mash. The hydrolysis may be carried out by acid treatment or enzymatically by α-amylase. Acid hydrolysis is used on a limited basis.

Enzymatic liquefaction is typically carried out as a three-step hot slurry process. The slurry is heated to between 60-95° C. (preferably 77-86° C., 80-85° C., and 83-85° C.) and the enzyme(s) is (are) added. Then the slurry is jet-cooked at between 95-140° C., preferably 105-125° C., cooled to 60-95° C. and more enzyme(s) is (are) added to obtain the final hydrolysis. The liquefaction process is carried out at pH 4.0-6.5, typically at a pH between 5 and 6.

The slurry may be incubated with an α-amylase and, optionally, a phytase (discussed herein) and incubated for 5 minutes to 2 hours, at a temperature range of 60 to 75° C. In a further liquefaction step, the incubated or pretreated starch-containing material may be exposed to an increase in temperature such as 0 to 45° C. above the starch gelatinization temperature of the starch-containing material (e.g., 70° C. to 120° C., 70° C. to 110° C., and 70° C. to 90° C.) for a period of time of 2 minutes to 6 hours (e.g., 2 minutes to 4 hrs, 90 minutes, 140 minutes and 90 to 140 minutes) at a pH of about 4.0 to 5.5 more preferably between 1 hour to 2 hours. The temperature can be increased by a conventional high temperature jet cooking system for a short period of time for example for 1 to 15 minutes. Then the starch maybe further hydrolyzed at a temperature ranging from 75° C. to 95° C., (e.g., 80° C. to 90° C. and 80° C. to 85° C.) for a period of 15 to 150 minutes (e.g., 30 to 120 minutes). The pH may not be adjusted during these process steps and the pH of the liquefied mash is in the range of pH 4.0 to pH 5.8 (e.g., pH 4.5 to 5.8, pH 4.8 to 5.4, and pH 5.0 to 5.2). In some embodiments, a second dose of thermostable a amylase will be added to the secondary liquefaction step, but in other embodiments there will not be an additional dosage of α-amylase.

The incubation and liquefaction steps according to the invention may be followed by saccharification and fermentation steps.

c. Fermentation

The fermentable sugars obtained during the liquefaction process steps may be used to produce alcohol, particularly ethanol, via microbial fermentation. The organism used in fermentations will depend on the desired end-product.

Typically if ethanol is the desired end product yeast will be used as the fermenting organism. In some preferred embodiments, the ethanol-producing microorganism is a yeast and specifically Saccharomyces such as strains of S. cerevisiae (U.S. Pat. No. 4,316,956). A variety of S. cerevisiae are commercially available and these include but are not limited to FALI (Fleischmann's Yeast), SUPERSTART (Alltech), FERMIOL (DSM Specialties), RED STAR (Lesaffre) and Angel alcohol yeast (Angel Yeast Company, China). The amount of starter yeast employed in the methods is an amount effective to produce a commercially significant amount of ethanol in a suitable amount of time, (e.g. to produce at least 10% ethanol from a substrate having between 25-40% DS in less than 72 hours). Yeast cells are generally supplied in amounts of 10⁴ to 10¹², and preferably from 10⁷ to 10¹⁰ viable yeast count per ml of fermentation broth. The fermentation will include in addition to a fermenting microorganisms (e.g. yeast), nutrients, optionally additional enzymes, including but not limited to phytases. The use of yeast in fermentation is well known and reference is made to THE ALCOHOL TEXTBOOK, K. JACQUES ET AL., EDS. 1999, NOTTINGHAM UNIVERSITY PRESS, UK. Yeast fermentation is usually performed for 24-96 hours, typically 35-60 hours. The temperature of fermentation is usually between 26-34° C., e.g., about 32° C., and the pH is bout 3-6, preferably around 4-5.

By using appropriate fermenting microorganisms, other end products may be obtained, including without limitation, glycerol, 1,3-propanediol, gluconate, 2-keto-D-gluconate, 2,5-diketo-D-gluconate, 2-keto-L-gulonic acid, succinic acid, lactic acid, amino acids and derivatives thereof. For example, when lactic acid is the desired end product, a Lactobacillus sp. (L. casei) may be used. When glycerol or 1,3-propanediol are the desired end-products E. coli may be used. When 2-keto-D-gluconate, 2,5-diketo-D-gluconate, and 2-keto-L-gulonic acid are the desired end products, Pantoea citrea may be used.

d. Saccharification and SSF

Liquefied starch-containing material is saccharified in the presence of saccharifying enzymes such as glucoamylases. The saccharification process may last for 12 hours to 120 hours (e.g. 12 to 90 hours, 12 to 60 hours and 12 to 48 hours). However, it is common to perform a pre-saccharification step for about 30 minutes to 2 hours (e.g., 30 to 90 minutes) in a temperature range of 30 to 65° C., typically above 50° C. and often around 60° C., which is followed by a complete saccharification during fermentation. This latter step may be referred to as simultaneous saccharification and fermentation (SSF). SSF is common in ethanol production, where the saccharifying enzymes and fermenting organisms (e.g., yeast) are added together and then carried out at a temperature of 30° C. to 40° C. and at a pH between 4.2-4.8, preferably pH 4.5.

Fermentable sugars (e.g., dextrins, monosaccharides, particularly glucose) are produced from enzymatic saccarification. These fermentable sugars may be further purified and/or converted to useful sugar products. In addition the sugars may be used as a fermentation feedstock in a microbial fermentation process for producing end-products, such as alcohol (e.g., ethanol and butanol), organic acids (e.g., succinic acid and lactic acid), sugar alcohols (e.g., glycerol), ascorbic acid intermediates (e.g., gluconate, 2-keto-D-gluconate, 2,5-diketo-D-gluconate, and 2-keto-L-gulonic acid), amino acids (e.g., lysine), proteins (e.g., antibodies and fragment thereof).

e. Distillation

Optionally, following fermentation, alcohol (e.g., ethanol) may be recovered by distillation. The yield of ethanol is typically at least 8%, at least 10%, at least 12%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18% (v/v), and is some cases, at least 19%, at least 20%, at least 21%, at least 22%, and even at least 23% (v/v). The ethanol obtained may be used as, for example, fuel ethanol, drinking ethanol, i.e., potable neutral spirits, or industrial ethanol.

f. By-Products

Left over from the fermentation process is the spent grain, which is typically used in animal feed either in liquid form or dried. The spent grain may take the form of so-called “fermentation co-products” such as distillers dried grains (DDG) and distiller's dried grain plus solubles (DDGS), which may also be used in animal feed.

3. Beer Making

The present variant α-amylases may be useful in a beer-making process. Typically, the α-amylases are added during the mashing process, where their advantages, in terms of stability, specific activity, and the like, are realized as in the case of starch conversion.

4. Use of Variant α-Amylases in Combination with a Other Enzymes

In all aspects of liquefaction, saccharification, SSF, and carbohydrate processing, generally, the present variant α-amylase polypeptides can be used in combination with one or more addition enzymes, for example, an additional α-amylase, a glucoamylase, an iso-amylase, a β-amylase, a maltogenic amylase, a protease, a lipase, a peroxidase, an esterase, an oxidase, a pectinase, a pectine lyase, a cutinase, a laccase, and/or a phytase. Many of these enzymes are described in more detail with respect to cleaning applications.

Phytases are enzymes capable of breaking down phytic acid (phytate) found in grains and oil seeds. Phytate, as well as intermediates in it degredation, are believed to destabilize or otherwise adversely affect α-amylases, thereby reducing their efficiency.

Phytases that can be used in combination with variant α-amylases are capable of hydrolyzing phytic acid under the defined conditions of the incubation and liquefaction steps. In some embodiments, the phytase is capable of liberating at least one inorganic phosphate from an inositol hexaphosphate (phytic acid). Phytases can be grouped according to their preference for a specific position of the phosphate ester group on the phytate molecule at which hydrolysis is initiated, (e.g., as 3-phytases (EC 3.1.3.8) or as 6-phytases (EC 3.1.3.26)). A typical example of phytase is myo-inositol-hexakiphosphate-3-phosphohydrolase.

Phytases can be obtained from microorganisms such as fungal and bacterial organisms. Some of these microorganisms include e.g. Aspergillus (e.g., A. niger, A. terreus, A. ficum and A. fumigatus), Myceliophthora (M. thermophila), Talaromyces (T. thermophilus) Trichoderma spp (T. reesei). and Thermomyces (WO 99/49740). Also phytases are available from Penicillium species, e.g., P. hordei (ATCC No. 22053), P. piceum (ATCC No. 10519), or P. brevi-compactum (ATCC No. 48944). See, for example U.S. Pat. No. 6,475,762. In addition, phytases are available from Bacillus (e.g., B. subtilis, Pseudomonas, Peniophora, E. coli, Citrobacter, Enterbacter and Buttiauxella (see WO2006/043178).

Commercial phytases are available such as NATUPHOS® (BASF), RONOZYME® P (Novozymes A/S), PHZYME® (Danisco A/S, Diversa) and FINASE® (AB Enzymes). The method for determining microbial phytase activity and the definition of a phytase unit has been published by Engelen et al. (1994) J. AOAC Int. 77:760-764. The phytase may be a wild-type phytase, a variant or fragment thereof.

Exemplary phytases are derived from species of the bacterium Buttiauxiella. Buttiauxiella spp. includes B. agrestis, B. brennerae, B. ferragutiase, B. gaviniae, B. izardii, B. noackiae, and B. warmboldiae. Strains of Buttiauxella species are available from DSMZ, the German National Resource Center for Biological Material (Inhoffenstrabe 7B, 38124 Braunschweig, Del.). Buttiauxella sp. strain P1-29 deposited under accession number NCIMB 41248 is an example of a particularly useful strain from which a phytase may be obtained. The phytase may be BP-wild type, a variant thereof (such as BP-11) described in WO 06/043178, or a variant as described in U.S. Patent Pub. No. US20080220498, filed Mar. 6, 2007 (see, e.g., Table 1 and SEQ ID NO: 3).

The phytase may also be the BP-17 variant of Buttiauxiella phytase, having the amino acid sequence of SEQ ID NO: 19, shown below, or a phytase having at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% and even at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 19.

(SEQ ID NO: 19) NDTPASGYQV EKVVILSRHG VRAPTKMTQT MRDVTPNTWP EWPVKLGYIT PRGEHLISLM GGFYRQKFQQ QGILSQGSCP TPNSIYVWAD VDQRTLKTGE AFLAGLAPQC GLTIHHQQNL EKADPLFHPV KAGTCSMDKT QVQQAVEKEA QTPIDNLNQH YIPFLALMNT TLNFSTSAWC QKHSADKSCD LGLSMPSKLS IKDNGNKVAL DGAIGLSSTL AEIFLLEYAQ GMPQAAWGNI HSEQEWASLL KLHNVQFDLM ARTPYIARHN GTPLLQAISN ALNPNATESK LPDISPDNKI LFIAGHDTNI ANIAGMLNMR WTLPGQPDNT PPGGALVFER LADKSGKQYV SVSMVYQTLE QLRSQTPLSL NQPAGSVQLK IPGCNDQTAE GYCPLSTFTR VVSQSVEPGC QLQ

The amount (dosage) of phytase used in the incubation and/or liquefaction processes may be in the range of about 0.001 to 50 FTU/g ds, (e.g., in the range of about 0.01 to 25 FTU/g ds, about 0.01 to 15 FTU/g ds, about 0.01 to 10 FTU/g ds, about 0.05 to 15 FTU/g ds, and about 0.05 to 5.0 FTU/g.

5. Compositions for Grain Processing and Starch Conversion

One aspect of the present compositions and methods is a composition comprising one or more of the variant α-amylases for use in starch conversion, including general starch conversion, alcohol fermentation, beer making, and the like. Such compositions may include buffers, salts, minerals, stabilizers, preservatives, antimicrobial agents, dyes, fragrances, and the like, selected to protect the variant α-amylase(s) from premature degredation (including proteolysis), to prolong storage, improve appearance, to color-code the composition, and the like.

The compositions may further include additional enzymes relating to starch conversion, including, e.g., glucoamylases and phytases. Particular a glucoamylases are G1 or G2 AMG from Aspergillus niger, which is described in Boel et al. (1984) EMBO J. 3:1097-1102 or a variant, thereof, as described in WO 00/04136 or WO 01/04273), the AMG from Talaromyces emersonii, as described in WO 99/28448, or glucoamylase from Trichoderma reesei, as described in WO 06/060062.

B. Pulp and Paper Production

The present variant α-amylases may also be used in the production of lignocellulosic materials, such as pulp, paper and cardboard, from starch reinforced waste paper and cardboard, especially where repulping occurs at pH above 7 and where the amylases facilitate the disintegration of the waste material through degradation of the reinforcing starch. The variant α-amylases are especially useful in a process for producing a papermaking pulp from starch-coated printed-paper. The process may be performed as described in WO 95/14807, comprising the steps of: (a) disintegrating the paper to produce a pulp, (b) treating with a starch-degrading enzyme before, during or after step (a), and (c) separating ink particles from the pulp after steps (a) and (b).

The α-amylases may also be useful where enzymatically modified starch is used in papermaking together with alkaline fillers such as calcium carbonate, kaolin and clays. With the present variant α-amylases, it is possible to modify the starch in the presence of the filler thus allowing for a simpler integrated process.

C. Desizing of Textiles, Fabrics and Garments

The present variant α-amylases may also be useful in textile, fabric or garment desizing. In the textile processing industry, α-amylases are traditionally used as auxiliaries in the desizing process to facilitate the removal of starch-containing size, which has served as a protective coating on weft yams during weaving. Complete removal of the size coating after weaving is important to ensure optimum results in the subsequent processes, in which the fabric is scoured, bleached and dyed. Enzymatic starch breakdown is preferred because it does not involve any harmful effect on the fiber material.

To reduce processing cost and increase mill throughput, the desizing processing is sometimes combined with the scouring and bleaching steps. In such cases, non-enzymatic auxiliaries such as alkali or oxidation agents are used to break down the starch, because traditional α-amylases are not very compatible with high pH levels and bleaching agents. The non-enzymatic breakdown of the starch size leads to some fiber damage because of the rather aggressive chemicals used. Accordingly, it would be desirable to use variant α-amylases that offer improved performance in alkaline solutions. Such variants may be used alone or in combination with a cellulase when desizing cellulose-containing fabric or textile.

Desizing and bleaching processes are well known in the art. For instance, such processes are described in WO 95/21247, U.S. Pat. No. 4,643,736, EP 119,920 hereby incorporated by reference. A current commercially available products for desizing include OPTISIZE® FLEX from Genencor.

D. Cleaning and Detergent Compositions

The present variant α-amylases may be added to, and thus become a component of, a detergent composition. The detergent composition may be formulated as a hand or machine-laundry detergent, including a laundry additive suitable for pretreatment of stained fabrics and a rinse-added fabric softener composition. The detergent composition may also be formulated for hand or machine-dishwashing operations, or for use in general household hard surface cleaning operations. In general the properties of the variant α-amylase should be compatible with the selected detergent in terms of its pH and other enzymatic and non-enzymatic ingredients.

The detergent composition or additive may comprise one or more additional enzymes such as a protease, a lipase, a peroxidase, another amylolytic enzyme (e.g., another α-amylase), a glucoamylase, a maltogenic amylase, a CGTase and/or a cellulase mannanase (such as MANNASTAR™ from Danisco US Inc., Genencor Division)), a pectinase, a pectine lyase, a cutinase, and/or laccase, which are described in more detail, below:

Proteases: Suitable proteases may be derived from any organism, and include chemically modified or engineered variants. The protease may be a serine protease or a metallo protease, preferably an alkaline microbial protease or a trypsin-like protease. Examples of alkaline proteases are subtilisins, especially those derived from Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89/06279). Examples of trypsin-like pro-teases are trypsin (e.g., of porcine or bovine origin) and the Fusarium protease described in WO 89/06270 and WO 94/25583.

Examples of useful proteases are the variants described in WO98/23732, WO99/20770, WO 92/19729, WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants with substitutions in one or more of the following positions: 27, 36, 57, 76, 87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235 and 274.

Preferred commercially available protease enzymes include ALCALASE®, SAVINASE®, PRIMASE®, DURALASE®, ESPERASE®, and KANNASE® (from Novozymes A/S), MAXATASE®, MAXACAL, MAXAPEM®, PROPERASE®, PURAFECT®, PURAFECT OXP®, FN2®, FN3®, FN4® (Genencor International Inc.).

Lipases: Suitable lipases include those of bacterial or fungal origin, and include chemically modified or engineered variant. Examples of useful lipases include lipases from Humicola (synonym Thermomyces), e.g., from H. lanuginosa (T. lanuginosus) as described in EP 258 068 and EP 305 216 or from H. insolens as described in WO 96/13580, a Pseudomonas lipase, e.g., from P. alcaligenes or P. pseudoalcaligenes (EP 218 272), P. cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P. fluorescens, Pseudomonas sp. strain SD 705 (WO 95/06720 and WO 96/27002), P. wisconsinensis (WO 96/12012), a Bacillus lipase, e.g., from B. subtilis (Dartois et al. (1993), Biochemica et Biophysica Acta, 1131, 253-360), B. stearothermophilus (JP 64/744992) or B. pumilus (WO 91/16422). Other examples are lipase variants such as those described in WO 92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO 97/07202. Preferred commercially available lipase enzymes include LIPOLASE™ and LIPOLASE ULTRA™ (Novozymes A/S).

Amylases: One or more additional amylases may also be included. Suitable amylases (α and/or β) include those of bacterial or fungal origin, and include chemically modified or engineered variants. Amylases include, for example, α-amylases obtained from Bacillus, e.g., a special strain of B. licheniformis, described in more detail in GB 1,296,839. Examples of useful α-amylases are the variants described in WO 94/18314, WO 96/39528, WO 94/02597, WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants with substitutions in one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243, 264, 304, 305, 391, 408, and 444. Commercially available α-amylases are DURAMYL™, LIQUEZYME™ TERMAMY™, NATALASE™, FUNGAMYL™ and BAN™ (Novozymes A/S), RAPIDASE™ and PURASTAR™ (from Genencor).

Cellulases: Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Trichoderma, Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum disclosed in U.S. Pat. No. 4,435,307, U.S. Pat. No. 5,648,263, U.S. Pat. No. 5,691,178, U.S. Pat. No. 5,776,757 and WO 89/09259. The Trichoderma reesei cellulases are disclosed in U.S. Pat. No. 4,689,297, U.S. Pat. No. 5,814,501, U.S. Pat. No. 5,324,649, WO 92/06221 and WO 92/06165. Bacillus cellulases are disclosed in U.S. Pat. No. 6,562,612. Commercially available cellulases include CELLUZYME®, and CAREZYME® (Novozymes A/S), CLAZINASE®, and PURADAX HA® (Genencor International Inc.), and KAC-500(B)® (Kao Corporation).

Peroxidases/Oxidases: Suitable peroxidases/oxidases include those of plant, bacterial or fungal origin, and include chemically modified or engineered variants. Examples of useful peroxidases include peroxidases from Coprinus, e.g., from C. cinereus, and variants thereof as those described in WO 93/24618, WO 95/10602, and WO 98/15257. Commercially available peroxidases include GUARDZYME® (Novozymes A/S).

The detergent enzyme(s) may be included in a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all of these enzymes. A detergent additive can be formulated as a liquid, a slurry, a bar, a tablet, a powder, a granule, a paste, etc. Exemplary detergent additive formulations are non-dusting granulates and stabilized liquids or slurries. A liquid detergent may be aqueous, typically containing up to 70% water and 0-30% organic solvent, or non-aqueous.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat. Nos. 4,106,991 and 4,661,452, and may optionally be coated by methods known in the art. Examples of waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 20000; ethoxylated nonyl-phenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in GB 1483591. Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods. Protected enzymes may be prepared according to the method disclosed in EP 238,216.

The detergent composition typically comprises one or more surfactants, which may be non-ionic (including semi-polar), anionic, cationic, and/or zwitterionic. The surfactants are typically present at a level of from 0.1% to 60% by weight. Exemplary detergent compositions include from about 1% to about 40% of an anionic surfactant such as linear alkylbenzenesulfonate, α-olefinsulfonate, alkyl sulfate (fatty alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate, α-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid or soap, and/or from about 0.2% to about 40% of a non-ionic surfactant such as alcohol ethoxylate, nonyl-phenol ethoxylate, alkylpolyglycoside, alkyldimethylamine-oxide, ethoxylated fatty acid monoethanol-amide, fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives of glucosamine (“glucamides”).

The detergent composition may include 0-65% detergent builder or complexing agent such as zeolite, diphosphate, tripho-sphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraacetic acid, diethylenetri-aminepen-taacetic acid, alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst). The detergent composition may include one or more polymers, such as carboxymethylcellulose, poly(vinyl-pyrrolidone), poly(ethylene glycol), poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole), polycarboxylates such as polyacrylates, maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid co-polymers.

The detergent may contain a bleaching system, which may include a H₂O₂ source such as perborate or percarbonate which may be combined with a peracid-forming bleach activator such as tetraacetylethylenediamine or nonanoyloxyben-zenesul-fonate. Alternatively, the bleaching system may comprise peroxyacids of, e.g., the amide, imide, or sulfone type.

The enzyme(s) of the detergent composition may be stabilized using conventional stabilizing agents, e.g., a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, and the composition may be formulated as described in, e.g., WO 92/19709 and WO 92/19708.

The detergent composition may also contain other conventional detergent composition ingredients, such as fabric conditioners, clays, foam boosters, suds suppressors, anti-corrosion agents, soil-suspending agents, anti-soil re-deposition agents, dyes, bactericides, optical brighteners, hydrotropes, tarnish inhibitors, or perfumes.

The variant α-amylase should be present in an effective amount, which can readily be determined using the assays described, herein. As a starting point, it is contemplated that one (or more) variant α-amylases be added in an amount corresponding to 0.01-100 mg of enzyme protein per liter of wash liquor, and preferably about 0.1-1 mg of enzyme protein per liter of wash liquor. An exemplary amount is about 0.055 mg of enzyme protein per liter of wash liquor.

Exemplary dish washing detergent compositions, include the following:

1) POWDER AUTOMATIC DISHWASHING COMPOSITION Nonionic surfactant 0.4-2.5% Sodium metasilicate 0-20% Sodium disilicate 3-20% Sodium triphosphate 20-40% Sodium carbonate 0-20% Sodium perborate 2-9% Tetraacetyl ethylene diamine (TAED) 1-4% Sodium sulphate 5-33% Enzymes 0.0001-0.1% 2) POWDER AUTOMATIC DISHWASHING COMPOSITION Nonionic surfactant 1-2% (e.g., alcohol ethoxylate) Sodium disilicate 2-30% Sodium carbonate 10-50% Sodium phosphonate 0-5% Trisodium citrate dihydrate 9-30% Nitrilotrisodium acetate (NTA) 0-20% Sodium perborate monohydrate 5-10% Tetraacetyl ethylene diamine (TAED) 1-2% Polyacrylate polymer 6-25% (e.g., maleic acid/acrylic acid copolymer) Enzymes 0.0001-0.1% Perfume 0.1-0.5% Water 5-10% 3) POWDER AUTOMATIC DISHWASHING COMPOSITION Nonionic surfactant 0.5-2.0% Sodium disilicate 25-40% Sodium citrate 30-55% Sodium carbonate 0-29% Sodium bicarbonate 0-20% Sodium perborate monohydrate 0-15% Tetraacetyl ethylene diamine (TAED) 0-6% Maleic acid/acrylic acid copolymer 0-5% Clay 1-3% Polyamino acids 0-20% Sodium polyacrylate 0-8% Enzymes 0.0001-0.1% 4) POWDER AUTOMATIC DISHWASHING COMPOSITION Nonionic surfactant 1-2% Zeolite MAP 15-42% Sodium disilicate 30-34% Sodium citrate 0-12% Sodium carbonate 0-20% Sodium perborate monohydrate 7-15% Tetraacetyl ethylene 0-3% diamine (TAED) Polymer 0-4% Maleic acid/acrylic acid copolymer 0-5% Organic phosphonate 0-4% Clay 1-2% Enzymes 0.0001-0.1% Sodium sulphate Balance 5) POWDER AUTOMATIC DISHWASHING COMPOSITION Nonionic surfactant 1-7% Sodium disilicate 18-30% Trisodium citrate 10-24% Sodium carbonate 12-20% Monopersulphate 15-21% (2 KHSO₅•KHSO₄•K₂SO₄) Bleach stabilizer 0.1-2% Maleic acid/acrylic acid copolymer 0-6% Diethylene triamine pentaacetate, 0-2.5% pentasodium salt Enzymes 0.0001-0.1% Sodium sulphate, water Balance 6) POWDER AND LIQUID DISHWASHING COMPOSITION WITH CLEANING SURFACTANT SYSTEM Nonionic surfactant 0-1.5% Octadecyl dimethylamine N-oxide 0-5% dihydrate 80:20 wt. C18/C16 blend of  0-4% octadecyl dimethylamine N-oxide  dihydrate and hexadecyldimethyl amine N-oxide dihydrate  70:30 wt. C18/C16 blend of   0-5% octadecyl bis (hydroxyethyl) amine N-oxide anhydrous and hexadecyl bis (hydroxyethyl) amine N-oxide anhydrous C₁₃-C₁₅ alkyl ethoxysulfate with an  0-10% average degree of ethoxylation of 3 C₁₂-C₁₅ alkyl ethoxysulfate with an  0-5% average degree of ethoxylation of 3 C₁₃-C₁₅ ethoxylated alcohol with an  0-5% average degree of ethoxylation of  12 A blend of C₁₂-C₁₅ ethoxylated   0-6.5% alcohols with an average degree    of ethoxylation of 9 A blend of C₁₃-C₁₅ ethoxylated   0-4% alcohols with an average degree    of ethoxylation of 30 Sodium disilicate 0-33% Sodium tripolyphosphate 0-46% Sodium citrate 0-28% Citric acid 0-29% Sodium carbonate 0-20% Sodium perborate monohydrate 0-11.5% Tetraacetyl ethylene diamine (TAED) 0-4% Maleic acid/acrylic acid copolymer 0-7.5% Sodium sulphate 0-12.5% Enzymes 0.0001-0.1% 7) NON-AQUEOUS LIQUID AUTOMATIC DISHWASHING  COMPOSITION Liquid nonionic surfactant 2.0-10.0% (e.g. alcohol ethoxylates) Alkali metal silicate 3.0-15.0% Alkali metal phosphate 20.0-40.0% Liquid carrier selected from higher 25.0-45.0% glycols, polyglycols, polyoxides,  glycolethers Stabilizer (e.g. a partial ester of  0.5-7.0% phosphoric acid and a C₁₆-C₁₈  alkanol) Foam suppressor (e.g. silicone) 0-1.5% Enzymes 0.0001-0.1% 8) NON-AQUEOUS LIQUID DISHWASHING COMPOSITION Liquid nonionic surfactant 2.0-10.0% (e.g. alcohol ethoxylates) Sodium silicate 3.0-15.0% Alkali metal carbonate 7.0-20.0% Sodium citrate 0.0-1.5% Stabilizing system (e.g. mixtures   0.5-7.0% of finely divided silicone and low   molecular weight dialkyl polyglycol  ethers) Low molecule weight polyacrylate  5.0-15.0% polymer Clay gel thickener (e.g. bentonite) 0.0-10.0% Hydroxypropyl cellulose polymer 0.0-0.6% Enzymes 0.0001-0.1% Liquid carrier selected from higher  Balance lycols, polyglycols, polyoxides and  glycol ethers 9) THIXOTROPIC LIQUID AUTOMATIC DISHWASHING  COMPOSITION C₁₂-C₁₄ fatty acid 0-0.5% Block co-polymer surfactant 1.5-15.0% Sodium citrate 0-12% Sodium tripolyphosphate 0-15% Sodium carbonate 0-8% Aluminium tristearate 0-0.1% Sodium cumene sulphonate 0-1.7% Polyacrylate thickener 1.32-2.5% Sodium polyacrylate 2.4-6.0% Boric acid 0-4.0% Sodium formate 0-0.45% Calcium formate 0-0.2% Sodium n-decydiphenyl oxide  0-4.0% disulphonate Monoethanol amine (MEA) 0-1.86% Sodium hydroxide (50%) 1.9-9.3% 1,2-Propanediol 0-9.4% Enzymes 0.0001-0.1% Suds suppressor, dye, perfumes,  Balance water 10) LIQUID AUTOMATIC DISHWASHING COMPOSITION Alcohol ethoxylate 0-20% Fatty acid ester sulphonate 0-30% Sodium dodecyl sulphate 0-20% Alkyl polyglycoside 0-21% Oleic acid 0-10% Sodium disilicate monohydrate 18-33% Sodium citrate dihydrate 18-33% Sodium stearate 0-2.5% Sodium perborate monohydrate 0-13% Tetraacetyl ethylene diamine  0-8% (TAED) Maleic acid/acrylic acid copolymer 4-8% Enzymes 0.0001-0.1% 11) LIQUID AUTOMATIC DISHWASHING COMPOSITION CONTAINING PROTECTED BLEACH PARTICLES Sodium silicate 5-10% Tetrapotassium pyrophosphate 15-25% Sodium triphosphate 0-2% Potassium carbonate 4-8% Protected bleach particles, e.g.  5-10% chlorine Polymeric thickener 0.7-1.5% Potassium hydroxide 0-2% Enzymes 0.0001-0.1% Water Balance 12) Automatic dishwashing compositions as  described in 1), 2), 3),4), 6) and 10),  wherein perborate is replaced by percarbonate. 13) Automatic dishwashing compositions as  described in 1)-6) which additionally contain  a manganese catalyst. The manganese catalyst  may, e.g., be one of the compounds described in “Efficient manganese catalysts for low- temperature bleaching”, Nature 369, 1994,  pp. 637-639.

VII. Methods for Measuring α-Amylase Properties

This section describes basic assays for measuring properties of α-amylases. Additional assays are described in the Examples section.

A. Filter Screening Assays

The following assays may be used to screening of SPEZYME® Xtra-like α-amylase variants having altered stability at high or low pH and/or under Ca²⁺ depleted conditions compared to the parent enzyme and SPEZYME® Xtra-like α-amylase.

1. High pH Filter Assay

Bacillus libraries are plated on a sandwich of cellulose acetate (OE 67, Schleicher & Schuell, Dassel, Del.)—and nitrocellulose filters (Protran-Ba 85, Schleicher & Schuell, Dassel, Del.) on TY agar plates with 10 micro g/ml kanamycin at 37° C. for at least 21 hours. The cellulose acetate layer is located on the TY agar plate.

Each filter sandwich is specifically marked with a needle after plating, but before incubation in order to be able to localize positive variants on the filter and the nitrocellulose filter with bound variants is transferred to a container with glycin-NaOH buffer, pH 8.6-10.6 and incubated at room temperature (can be altered from 10-60° C.) for 15 min. The cellulose acetate filters with colonies are stored on the TY-plates at room temperature until use. After incubation, residual activity is detected on plates containing 1% agarose, 0.2% starch in glycin-NaOH buffer, pH 8.6-10.6. The assay plates with nitrocellulose filters are marked the same way as the filter sandwich and incubated for 2 hours at room temperature. After removal of the filters the assay plates are stained with 10% Lugol solution. Starch degrading variants are detected as white spots on dark blue background and then identified on the storage plates. Positive variants are rescreened twice under the same conditions as the first screen.

2. Low Calcium Filter Assay

Bacillus libraries are plated on a sandwich of cellulose acetate (OE 67, Schleicher & Schuell, Dassel, Del.)—and nitrocellulose filters (Protran-Ba 85, Schleicher & Schuell, Dassel, Del.) on TY agar plates with a relevant antibiotic, e.g., kanamycin or chloramphenicol, at 37° C. for at least 21 hours. The cellulose-acetate layer is located on the TY agar plate.

Each filter sandwich is specifically marked with a needle after plating, but before incubation in order to be able to localize positive variants on the filter and the nitrocellulose filter with bound variants is transferred to a container with carbonate/bicarbonate buffer pH 8.5-10 and with different EDTA concentrations (0.001 mM-100 mM). The filters are incubated at room temperature for 1 hour. The cellulose acetate filters with colonies are stored on the TY-plates at room temperature until use. After incubation, residual activity is detected on plates containing 1% agarose, 0.2% starch in carbonate/bicarbonate buffer pH 8.5-10. The assay plates with nitrocellulose filters are marked the same way as the filter sandwich and incubated for 2 hours at room temperature. After removal of the filters the assay plates are stained with 10% Lugol solution. Starch degrading variants are detected as white spots on dark blue background and then identified on the storage plates. Positive variants are rescreened twice under the same conditions as the first screen.

3. Low pH Filter Assay

Bacillus libraries are plated on a sandwich of cellulose acetate (OE 67, Schleicher & Schuell, Dassel, Del.)—and nitrocellulose filters (Protran-Ba 85, Schleicher & Schuell, Dasseli, Del.) on TY agar plates with 10 micro g/ml chloramphenicol at 37° C. for at least 21 hours. The cellulose acetate layer is located on the TY agar plate.

Each filter sandwich is specifically marked with a needle after plating, but before incubation in order to be able to localize positive variants on the filter, and the nitrocellulose filter with bound variants is transferred to a container with citrate buffer, pH 4.5 and incubated at 80° C. for 20 minutes (when screening for variants in the wild type backbone) or 85° C. for 60 minutes (when screening for variants of the parent α-amylase). The cellulose acetate filters with colonies are stored on the TY-plates at room temperature until use. After incubation, residual activity is detected on assay plates containing 1% agarose, 0.2% starch in citrate buffer, pH 6.0. The assay plates with nitrocellulose filters are marked the same way as the filter sandwich and incubated for 2 hours at 50° C. After removal of the filters the assay plates are stained with 10% Lugol solution. Starch degrading variants are detected as white spots on dark blue background and then identified on the storage plates. Positive variants are re-screened twice under the same conditions as the first screen.

3. Secondary Screening

Positive transformants after rescreening are picked from the storage plate and tested in a secondary plate assay. Positive transformants are grown for 22 hours at 37° C. in 5 ml LB+ chloramphenicol. The Bacillus culture of each positive transformant and as a control a clone expressing the corresponding backbone are incubated in citrate buffer, pH 4.5 at 90° C. and samples are taken at 0, 10, 20, 30, 40, 60 and 80 minutes. A 3 μL sample is spotted on an assay plate. The assay plate is stained with 10% Lugol solution. Improved variants are seen as variants with higher residual activity (detected as halos on the assay plate) than the backbone. The improved variants are determined by nucleotide sequencing.

B. Stability Assay of Unpurified Variants

The stability of the variants may be assayed as follows: Bacillus cultures expressing the variants to be analyzed are grown for 21 hours at 37° C. in 10 ml LB+ chloramphenicol. 800 μL culture is mixed with 200 μL citrate buffer, pH 4.5. A number of 70 μL aliquots corresponding to the number of sample time points are made in PCR tubes and incubated at 70° C. or 90° C. for various time points (typically 5, 10, 15, 20, and 30 minutes) in a PCR machine. The 0 min sample is not incubated at high temperature. Activity in the sample is measured by transferring 20 μL to 200 μL of the α-amylase PNP-G₇ substrate MPR3 ((Boehringer Mannheim Cat. no. 1660730) as described below under “Assays for α-Amylase Activity”. Results are plotted as percentage activity (relative to the 0 time point) versus time, or stated as percentage residual activity after incubation for a certain period of time.

C. Fermentation and Purification of α-Amylase Variants

A B. subtilis strain harboring the relevant expression plasmid may be fermented and purified as follows: The strain is streaked on a LB-agar plate with 10 μg/ml kanamycin from −80° C. stock, and grown overnight at 37° C. The colonies are transferred to 100 ml PS-1 media supplemented with 10 micro g/ml chloamphinicol in a 500 ml shaking flask. The culture is shaken at 37° C. at 270 rpm for 5 days.

Composition of PS-1 Medium:

Pearl sugar 100 g/l  Soy Bean Meal 40 g/l Na₂HPO₄, 12 H₂O 10 g/l Pluronic ™ PE 6100 0.1 g/l  CaCO₃  5 g/l

Cells and cell debris are removed from the fermentation broth by centrifugation at 4500 rpm in 20-25 minutes. Afterwards the supernatant is filtered to obtain a completely clear solution. The filtrate is concentrated and washed on a UF-filter (10,000 MW cut-off membrane) and the buffer is changed to 20 mM Acetate pH 5.5. The UF-filtrate is applied on an S-sepharose FF column and elution is carried out by step elution with 0.2 M NaCl in the same buffer. The eluate is dialysed against 10 mM Tris, pH 9.0 and applied on a Q-sepharose FF column and eluted with a linear gradient from 0-0.3 M NaCl over 6 column-volumes. The fractions that contain the activity (measured by the Phadebas assay) are pooled, pH was adjusted to pH 7.5 and remaining color was removed by a treatment with 0.5% w/vol. activated charcoal for 5 minutes.

D. Specific Activity Determination

The specific activity is determined using the PHADEBAS® assay (Pharmacia) as activity/mg enzyme. The manufactures instructions are followed (see also below under “Assay for α-Amylase Activity).

E. Determination of Isoelectric Point

The pI is determined by isoelectric focusing (e.g., Pharmacia, Ampholine, pH 3.5-9.3).

F. Stability Determination

The amylase stability may be measured using the method as follows:

The enzyme is incubated under the relevant conditions. Samples are taken at various time points, e.g., after 0, 5, 10, 15 and 30 minutes and diluted 25 times (same dilution for all taken samples) in assay buffer (50 mM Britton buffer pH 7.3) and the activity is measured using the Phadebas assay (Pharmacia) under standard conditions pH 7.3, 37° C.

The activity measured before incubation (0 minutes) is used as reference (100%). The decline in percent is calculated as a function of the incubation time. The table shows the residual activity after, e.g., 30 minutes of incubation.

G. Assays for α-Amylase Activity

1. PHADEBAS® Assay

α-amylase activity is determined by a method employing PHADEBAS® tablets as substrate. Phadebas tablets (PHADEBAS® Amylase Test, supplied by Pharmacia Diagnostic) contain a cross-linked insoluble blue-colored starch polymer, which has been mixed with bovine serum albumin and a buffer substance and tabletted.

For every single measurement one tablet is suspended in a tube containing 5 ml 50 mM Britton-Robinson buffer (50 mM acetic acid, 50 mM phosphoric add, 50 mM boricacid, 0.1 mM CaCl₂, pH adjusted to the value of interest with NaOH). The test is performed in a water bath at the temperature of interest. The α-amylase to be tested is diluted in x ml of 50 mM Britton-Robinson buffer. 1 ml of this α-amylase solution is added to the 5 ml 50 mM Britton-Robinson buffer. The starch is hydrolyzed by the α-amylase giving soluble blue fragments. The absorbance of the resulting blue solution, measured spectrophotometrically at 620 nm, is a function of the α-amylase activity.

It is important that the measured 620 nm absorbance after 10 or 15 minutes of incubation (testing time) is in the range of 0.2 to 2.0 absorbance units at 620 nm. In this absorbance range there is linearity between activity and absorbance (Lambert-Beer law). The dilution of the enzyme must therefore be adjusted to fit this criterion. Under a specified set of conditions (temp., pH, reaction time, buffer conditions) 1 mg of a given α-amylase will hydrolyze a certain amount of substrate and a blue color will be produced. The color intensity is measured at 620 nm. The measured absorbance is directly proportional to the specific activity (activity/mg of pure α-amylase protein) of the α-amylase in question under the given set of conditions.

2. Alternative Method

α-amylase activity is determined by a method employing the PNP-G₇ substrate. PNP-G₇ which is a abbreviation for p-nitrophenyl-α,D-maltoheptaoside is a blocked oligosaccharide which can be cleaved by an endo-amylase. Following the cleavage, the α-Glucosidase included in the kit digest the substrate to liberate a free PNP molecule which has a yellow color and thus can be measured by visible spectophometry at λ=405 nm (400-420 nm). Kits containing PNP-G₇ substrate and α-Glucosidase is manufactured by Boehringer-Mannheim (cat. No. 1054635).

To prepare the reagent solution 10 ml of substrate/buffer solution is added to 50 ml enzyme/buffer solution as recommended by the manufacturer. The assay is performed by transferring 20 μL sample to a 96 well microtitre plate and incubating at 25° C. 200 μL reagent solution pre-equilibrated to 25° C. is added. The solution is mixed and pre-incubated 1 minute and absorption is measured every 30 seconds over 4 minutes at OD 405 nm in an ELISA reader.

The slope of the time dependent absorption-curve is directly proportional to the activity of the α-amylase in question under the given set of conditions.

H. Determination of LAS Sensitivity

The variant is incubated with different concentrations of LAS (linear alkyl benzene sulphonate; Nansa 1169/P) for 10 minutes at 40° C. The residual activity is determined using the PHADEBAS® assay method or the alternative method employing the PNP-G₇ substrate. LAS is diluted in 0.1 M phosphate buffer pH 7.5. The following concentrations are used: 500 ppm, 250 ppm, 100 ppm, 50 ppm, 25 ppm, and 10 ppm on no LAS.

The variant is diluted in the different LAS buffers to concentration of 0.01-5 mg/l in a total volume of 10 ml and incubated for 10 minutes in a temperature controlled water bath. The incubation is stopped by transferring a small aliquot into cold assay buffer. It is important that during activity measurement the LAS concentration is below 1 ppm, in order not to affect the activity measurement.

Then the residual activity is determined in duplicate using the above mentioned PHADEBAS® assay or alternative method. The activity is measured after subtraction of the blank. The activity with no LAS is 100%.

G. Determination of Phytase Activity (FTU)

Phytase activity (FTU) is measured by the release of inorganic phosphate. The inorganic phosphate forms a yellow complex with acidic molybdate/vanadate reagent and the yellow complex is measured at a wavelength of 415 nm in a spectrophotometer and the released inorganic phosphate is quantified with a phosphate standard curve. One unit of phytase (FTU) is the amount of enzyme that releases 1 micromole of inorganic phosphate from phytate per minute under the reaction conditions given in the European Standard (CEN/TC 327,2005-TC327WI 003270XX).

H. Determination of Phytic Acid Content

To determine phytic acid content, phytic acid was extracted from sample by adjusting the pH of the 5% slurry (if it is dry sample) to pH 10 and then determined by an HPLC method using an ion exchange column. Phytic acid was eluted from the column using a NaOH gradient system. Phytic acid content in the liquid was then calculated by comparing to a phytic acid standard.

The present compositions and methods are described in further detail in the following examples which are not in any way intended to be limiting in scope. All references cited are herein specifically incorporated by reference for all that is described therein.

EXAMPLES

In the disclosure and experimental section which follows, the following abbreviations apply: wt % (weight percent); ° C. (degrees Centigrade); H₂O (water); dH₂O (deionized water); dIH₂O (deionized water, Milli-Q filtration); g or gm (grams); μg (micrograms); mg (milligrams); kg (kilograms); μL and μl (microliters); mL and ml (milliliters); mm (millimeters); μm (micrometer); M (molar); mM (millimolar); μM (micromolar); U (units); MW (molecular weight); sec (seconds); min(s) (minute/minutes); hr(s) (hour/hours); DO (dissolved oxygen); W/V (weight to volume); W/W (weight to weight); V/V (volume to volume); IKA (IKA Works Inc. 2635 North Chase Parkway SE, Wilmington, N.C.); Genencor (Danisco US Inc, Genencor Division, Palo Alto, Calif.); Ncm (Newton centimeter) and ETOH (ethanol). eq (equivalents); N (Normal); ds or DS (dry solids content), SAPU (spectrophotometric acid protease unit, wherein in 1 SAPU is the amount of protease enzyme activity that liberates one micromole of tyrosine per minute from a casein substrate under conditions of the assay) and GAU (glucoamylase unit, which is defined as the amount of enzyme that will produce 1 g of reducing sugar calculated as glucose per hour from a soluble starch substrate at pH 4.2 and 60° C.).

Example 1 Construction of Variants

Variants in the mature sequence of AmyS were constructed using a site directed approach. For example, position S242 variants were constructed as follows:

The template for mutagenesis was methylated pHPLT-AmyS (see FIG. 2) using dam-Methylase from New England Biolabs (Massachusetts). Degenerate primers (S242F(orward) and S242R(everse), given below) were synthesized and diluted to 10 μM at Operon (Huntsville, Ala.) with complementary forward and reverse sequences both containing a 5′ phosphate for ligation in the reaction. The sequence of the parent α-amylase is shown as SEQ ID NO: 2. Libraries were created with the Stratagene QUIK-CHANGE™ Multi-site kit (Stratagene, La Jolla Calif.) using oligonucleotide primers randomized with NN(G/C) at the target position. The selected amino acid (e.g., S242) was randomly replaced with all 19 possible alternatives.

S242 primers for mutagenesis: S242 F: SEQ ID NO: 17 5′-[Phos]GTCAAGCATATTAAGTTCNNSTTTTTTCCTGATTGGTTG- 3′ S242 R: SEQ ID NO: 18 5′-[Phos]CAACCAATCAGGAAAAAASNNGAACTTAATATGCTTGAC- 3′

The reaction was performed as follows:

Quik-Change Reaction:

The reaction consisted of 18 μL of sterile distilled H₂O, 2.5 μL of 10× buffer from the kit, 1 μL dNTPs from the kit, 1.25 μL of the forward primers (of 10 uM stock), 1.25 μL of the reverse primers (of 10 uM stock), 1 μL of pHPLT-AmyS plasmid DNA as template (˜70 ng), and 1 μL of the enzyme blend from the kit for a total of 26.5 μL.

Cycling Conditions:

The cycling conditions were 95° C. for 1 min once, then 95° C. for 1 min, 55° C. for 1 min, 65° C. for 10 min for 25 cycles. One μL Dpn I (10 U/μL) was added to the Multi-site Quik-Change reaction mixture and incubated at 37° C. for 18 hours and then another 0.5 μl was added for an additional 3 hours.

One μL of DpnI digested reaction was used as template for rolling circle amplification with the Templiphi amplification kit (Amersham Biosciences, Piscataway, N.J.) and the reaction was performed according to the Amersham protocol. One μL of rolling circle DNA was transformed into 100 μL of Bacillus subtilis competent cells (two protease deleted B. subtilis strain (ΔaprE, ΔnprE, amyE::xylRPxylAcomK-phleo)) and shaken at 37° C. for 1 hour. The entire transformation was next plated on LA+10 ppm Neo+1% insoluble starch plates (25 μL one plate, 75 μL on another plate) and incubated overnight at 37° C. 96 transformants were picked into 150 μL of LB+10 ppm Neo in a micro-titer plate and grown overnight at 37° C. The overnight plate was stamped onto a large LA+10 ppm Neo+1% insoluble starch plate with a 96-pin replicating tool and submitted to Quintara Biosciences (Berkeley, Calif., USA) for colony PCR and sequencing.

After variant sequences were determined, the variants were picked into a 96-well micro-titer plates containing 125 μL of LB+10 ppm Neo, arraying the variants into a quad format with controls. The arrayed micro-titer plate was grown for 6 hours at 37° C. and 250 rpm. Using a replicating tool (Enzyscreen, Leiden, The Netherlands) the micro-titer culture plate was used to inoculate a new micro-titer plate (micro-titer plate and plate lids from Enzyscreen, Leiden, The Netherlands) containing 150 μL of MBD medium for protein expression (G. Vogtentanz et al, A Bacillus subtilis fusion protein system to produce soybean Bowman-Birk protease inhibitor, Prot. Expr. & Purif., 55 (2007) 40-52) and supplemented with 5 mM CaCl₂ for protein expression. Expression plates were grown for 64 hours at 37° C., 250 rpm, and 70% humidity. Expression cultures were next filtered through a micro-filter plate (0.22 um, Millipore, Billerica, Mass.) and screened for improved thermostability (see Example 3).

AmyS Libraries

Site evaluation libraries were made for the following AmyS variants:

P17, D19, T21, N28, S51, G72, V74, A82, Q86, Q89, A93, W115, D117, P123, S124, D125, N127, I130, 6132, Q135, P145, 6146, G148A, S153A, Y159, W166, S169, K171, R179, G180, I181, G182, K183, W187, 6194, P209, N224, S242, P245, 6256, D269, N271, T278, N281, G302, A304, R308, T321, Q358, P378, S382, K383, T398, H405, T417, E418, P420, G421, P432, W437, Q443, G446, G454, S457, T459, T461, S464, G474, R483.

Example 2 Expression, Purification, and Characterization of Variants

Colonies were streaked from the microtiter plates from Example land put onto starch plates with 10 ppm Neomycin. The plates were incubated overnight at 37° C. and singles colonies were picked and used to inoculate shake flasks (250 mL with 25 mL media) containing media (see below) and 20 ppm Neomycin. These were grown up at 37° C., 275 rpm, for about 8 hrs (until an OD (600 nm) of 2.0 was reached). Whereupon the culture broths were mixed with 50% glycerol at 2:1 ratio, put into individually labeled culture vials and frozen at −80° C. It was from these glycerol stocks that subsequent production of the selected amylases were made.

Fermentations for amylases were carried out in 500 mL shake flasks grown at 37° C. for 60 hours in minimal MOPS culture medium (Neidhardt et al., J. Bacteriol. (1974) 119(3):736-747) with 1% (w/v) Soytone. Enzymes were purified from the fermentation broth using hydrophobic interaction chromatography. In brief, the broth were concentrated 10-fold then diluted back with 50 mM MES, 2 mM CaCl₂, pH 6.8 with 1M ammonium sulfate and sterile filtered using glass fiber filter. Samples were then load onto phenyl sepharose FF high density column (20×95 mm; Amersham, GE Healthcare Bio-Sciences, Sweden) pre-equilibrated with the same buffer. Non-amlyase proteins were washed off with 10 column volumes of the same buffer without ammonium sulfate followed by 5 column volumes of water. Finally, enzymes of interest were eluted with 50 mM MES, 2 mM CaCl₂, pH 6.8 containing 40% propylene glycol.

Protein concentrations were determined either by a standard quantitative SDS page gel densitometry method or by an activity assay using a standard amylase assay kit from Megazyme (Wicklow, Ireland). Assays were converted using a standard curve generated using purified amylase (Bacillus 707 amylase; SEQ ID NO: 6).

Example 3 Determination of Altered Properties: Thermal Stress

This example shows that the variants described herein may have an altered property relative to the parent α-amylase. A high throughput thermal stability screen of G. stearothermophilus α-amylase (AmyS) variants was carried out.

Heat stress conditions were investigated and chosen such that after the heat stress the starting wild-type enzyme showed approximately 40% of its unstressed activity (i.e., activity after heat stress/activity before heat stress was approximately 0.4). Libraries of mutants were screened in quadruplicate, and potential winners were identified as those that showed residual activity after heat stress that was at least two standard deviations more than the average residual activity of the starting wildtype enzyme.

Amylase expression was approximately 100 ppm in the culture supernatants of the expression plates. After 60-65 hours of growth at 37° C. in a humidified shaker (250 rpm and 70% relative humidity), the culture supernatants were clarified to remove cellular material using filter plates. The clarified supernatants were diluted 10-fold into buffer containing 50 mM NaOAc/2.6 mM CaCl₂/0.002% Tween-20, pH 5.8., to a final concentration of approximately 10 ppm. One aliquot of the supernatant was further diluted to 0.02 ppm, and activity of the enzyme variants were determined as described below using a fluorescently-labeled corn starch substrate. A second aliquot of the supernatant was subjected to a 30 minute heat stress at 95° C. in a thermocycler before being diluted to 0.02 ppm in 50 mM NaOAc/2.6 mM CaCl₂/0.002% Tween-20, pH 5.8 and assayed for residual activity using the same fluorescent substrate and assay described below.

Amylase activity was determined using the amylase EnzCheck assay essentially as described by the manufacturer (Invitrogen, San Diego Calif.). Final concentration of the amylase in the assay was approximately 0.02 ppm. Assay buffer was 50 mM NaOAc/2.6 mM CaCl₂/0.002% Tween-20, pH 5.8. The substrate was BODIPY fluorescence dye conjugated 100 μg/mL DQ™ starch from corn (Invitrogen—Eugene, Oreg.). Increased fluorescence, indicating amylase activity, was measured using a Spectomax M2 (Molecular Devices, Sunnyvale, Calif.). The reaction was monitored at room temperature for 5 minutes with the instrument recording in kinetic mode. Excitation wavelength was 485 nm; emission was monitored at 520 nm with a cutoff filter at 515 nm.

The wild type AmyS (SPEZYME® Xtra) showed 33-43% residual activity after being subject to thermal stress for 30 minutes at 95° C. AmyS variants, S242A and S242Q, retained 55-65% and 70-80% residual activities, respectively, following the same thermal stress conditions. See FIG. 3 and Table 3-1. These residual activity measurements indicate the two variants are more thermostable than the wild type a amylase. Some variants were missing from the libraries and are indicated by position letter within parenthesis marks. In its place, wild type (SPEZYME® Xtra) was placed instead; (WT) indicates the wild type was placed instead. Each plate includes SPEZYME® Xtra (labeled Z) as a control.

TABLE 3-1 Percent residual activities of each variant samples. Variants % Residual Activity Average Stdev % CV A 60.6 59.8 56.5 64.6 60.4 3.3 5 C 38.1 35.6 28.3 34.5 34.1 4.2 12 D 50.6 42.9 45.0 48.7 46.8 3.5 7 (E) (WT) 45.3 38.6 39.5 40.7 41.0 3.0 7 (F) (WT) 40.5 40.2 41.2 38.9 40.2 1.0 2 G 36.4 35.7 44.8 36.7 38.4 4.3 11 (H) (WT) 34.9 36.9 37.0 42.1 37.7 3.0 8 I 20.9 26.7 27.5 17.2 23.1 4.9 21 K 22.6 21.5 19.3 24.5 22.0 2.2 10 L 34.9 30.7 34.5 30.7 32.7 2.3 7 M 35.3 37.3 38.3 41.3 38.1 2.5 7 (N) (WT) 43.9 43.2 46.0 42.2 43.8 1.6 4 (P) (WT) 33.8 35.6 40.2 37.4 36.8 2.7 7 Q 80.6 71.0 75.9 71.5 74.8 4.5 6 R 9.6 4.5 6.1 5.4 6.4 2.2 35 (S) (WT) 38.6 39.9 37.2 37.3 38.3 1.3 3 T 36.8 31.5 35.1 27.8 32.8 4.0 12 V 25.0 24.7 25.0 22.9 24.4 1.0 4 (W) (WT) 32.7 37.5 36.3 38.8 36.3 2.6 7 (Y) (WT) 37.1 42.6 46.0 38.6 41.1 4.0 10 Z (Xtra) 38.8 41.5 42.5 32.7 38.9 4.4 11

Example 4 Determination of Altered Properties by DSC

SPEZYME® Xtra, S242A and S242Q were purified from shake flask fermentation broth (see Example 2) using hydrophobic interaction chromatography. The protein was eluted from the column in purified form using 50 mM MES, pH 6.8, containing 40% propylene glycol and 2 mM CaCl₂.

Excessive heat capacity curves were measured using an ultrasensitive scanning high-throughput microcalorimeter, VP-Cap DSC (MicroCal, Inc., Northampton, Mass.). The standard procedure for DSC measurements and the theory of the technique is previously published (Freire, E. (1995) Differential Scanning calorimetry Methods. Mol. Biol. 41, 191-218). Approximately 500 pt of 0.5 mg/ml wild type Bacillus stearothermophilus α-amylase or variant S242S and S242Q (in the absence and presence of 2 mM calcium chloride) were scanned over 30-120° C. temperature range. The same sample was then re-scanned to check the reversibility of the process. For α-amylase the thermal unfolding process was irreversible. The buffer used was 10 mM sodium acetate, pH 5.5. A 200° C./hr scan rate was used to minimize any artifacts that may result from aggregation. The thermal midpoint (Tm) of the DSC curves was used as an indicator of the thermal stability. Table 4-1 shows the thermal melting points for the amylase proteins tested. The thermal melting curves and the melting points for the wild type and amylase variants are shown in FIG. 5.

The thermal unfolding for the amylase variants S242A and S242Q in the absence and presence of 2 mM calcium chloride show considerable increase in the melting points for the variants when compared to that for the wild type. In the absence of added calcium chloride, the wild type amylase has a thermal melting point of 100.8° C. whilst the Tm's for S242A and S242Q are 106.5° C. and 110.1° C., respectively. Thus, the substitution of 5242 with A results in an increase in the Tm of 5.7° C., and the substitution of S242 with Q results in an increase in the Tm of 9.3° C.

In the presence of 2 mM calcium chloride, the wild type amylase characterized has a thermal melting point of 106.8° C. whilst the Tm's for S242A and S242Q are 111.8° C. and 113.8° C., respectively. Thus, in the presence of 2 mM calcium chloride all three proteins displayed increased Tm values. The increase in Tm for wild type and the S242A variants was 6° C. and 5.3° C., respectively. The increase in Tm for the S242Q variants was 3.7° C. This suggests that the S242Q variants is stabilized less by calcium or is less dependent on calcium for stability. The increase in the Tm of the S242A and S242Q relative to wild type in the presence of calcium chloride was 5° C. and 3° C., respectively. This suggests that the thermodynamic properties of the variants differ from those of SPEZYME® Xtra, and is consistent with its enhanced performance in application studies (see Example 5).

TABLE 4-1 Tm (no Ca₂ ⁺) Tm (w/2 mM Ca₂ ⁺) SPEZYME ® Xtra 100.8 106.8 S242A 106.5 111.8 S242Q 110.1 113.8

Example 5 Activity Profiles

This example shows that the tested variants have altered activity profiles relative not only to the parent α-amylase but also to an industry standard. Protein determinations were made on purified or plate samples. All experimental variants and standard α-amylases were dosed on equal protein concentrations.

Either plate or purified variants were diluted down to approximately 20 ppm using pH 5.6 malic acid buffer. The substrate consisted of 15% corn starch in the same 50 mM Malic acid buffer, pH 5.6. 400 μL of the starch suspension was equilibrated to 70° C. for 2.5 minutes. Then 7 μL of the diluted enzyme was quickly added to the equilibrated starch (final protein concentration of around 0.36 ppm). The reaction mix was then put into a pre-heated 85° C. shaking heating block and mixed at 300 rpm. At predetermined time intervals the reactions were quenched with 50 μL of 125 mM NaOH. The reaction tubes were then spun and the supernatent was diluted 10-fold into 10 mM NaOH, to be analyzed for DP profile by HPAEC-PAD.

Reactions were set up for 4, 10 and 20 minutes. Total area from DP2 to the end of the HPLC run was integrated and the area was divided by the total protein and reaction time.

The 4-minute reaction provides an indication of how quickly the enzyme begins to break down the substrate; the 10-minute reaction provides an indication of the enzyme's thermal activity, and the 20-minute reaction provides an indication of the enzyme's thermal stability. The results are provided in FIGS. 7 and 8.

Example 6 Liquefaction in the Viscometer

This example shows that the S242A and S242Q variants of Example 3 that had altered residual activity relative to the wild-type parent also have altered performance relative to the parent α-amylase. The variant α-amylases of Example 2 were purified and characterized for total protein and specific activity before its test in the application.

Viscosity reduction of corn flour due to the action of the α-amylase was monitored using a HAAKE Viscotester 550 instrument. The substrate slurry is made up fresh daily in batch mode with 30% corn flour dry solids. The pH was adjusted to 5.8 using sulfuric acid. 50 g of the slurry (15 g dry solids) is weighed out and pre-incubated, with stirring, for 10 minutes to warm up to 70° C. Upon α amylase addition the temperature is immediately ramped up from 70° C. to 85° C. with a rotation speed of 75. Once the temperature of the slurry and enzyme mixture reaches 85° C., its temperature is held constant and viscosity is monitored for an additional 30 minutes. The viscosity was measured throughout the run and is reported in μNm. Wildtype AmyS, S242A, and S242Q were all dosed at equal protein concentrations (20 or 30 μg/50 g of corn flour slurry).

The viscometer application test resulted in both AmyS variants, S242A and S242Q, having better performance than the benchmark α amylases—LIQUOZYME® SC and SPEZYME® Xtra. Both variants exhibit the low peak viscosity characteristic of SPEZYME® Xtra and low final viscosity of LIQUOZYME® SC. When loaded at the lower concentration of 20 μg total protein, the differences of lower peak viscosities of the variants compared to that of LIQUOZYME® SC are further enhanced. See FIGS. 9, 10 and 11.

Example 7 Liquefaction in a Jet Cooker

Whole ground corn was slurried to a 32% (dry solids corn) slurry by using a 70:30 ratio of water to thin stillage. The slurry pH was adjusted to pH 5.8 with 10 N NaOH. The slurry was heated to 70° C. (158° F.) using water and steam in a jacketed kettle. The liquefaction enzymes (SPEZYME® Xtra, LIQUOZYME® SC, or S242Q) were added and the slurry was heated to 85° C. (185° F.) over approximately 10 minutes. After an additional 10 minutes of incubation at 85° C., the slurry was passed through a jet-cooker maintained at 107° C. (225° F.) with a 3-minute hold time using a large pilot plant jet (equipped with an M103 hydro-heater from Hydro-thermal Corp., Waukesha, Wisconsin). The liquefact was collected from the jet and placed in an 85° C. water bath. A second dose of liquefaction enzyme was added post-jet. The liquefact was continuously stirred and held at 85° C. for 90 minutes. Samples were collected at 0, 30, 60 and 90 minutes. All samples were tested post-jet for DE (using the Schoorls method; method available upon request), and for viscosity (Brookfield-type viscometer (Lab-line Instruments Inc. of Melrose Park, Ill.) spindle 3 at 20 rpm). Dosing of liquefaction enzymes pre- and post-jet are indicated in the following Figures as “X+Y” where X represents the number of units of enzyme added before the jet, and Y represents the number of units added to the liquefact after it passes through the jet cooker. Results are shown in FIGS. 12 and 13.

Example 8 Effect of Removal of Phytic Acid Inhibition on a Amylase Thermostability

In this example, the effect of the removal of phytic acid inhibition on the thermostability of liquefying thermostable α amylases was examined.

A. No Jet Cooking (Single Enzyme Dose)

A slurry of whole ground corn (obtained from Badger State Ethanol, Monroe, Wis., USA) was made with water containing 30% v/v thin stillage to a final concentration of about 32% ds. Corn solids were prepared in a jacked kettle. The slurry was mixed well and the pH of the slurry was measured (pH 5.2) and was used without further pH adjustment. This slurry was mixed in a jacketed kettle and brought up to the pretreatment temperature of 70° C. Just prior to reaching 70° C., the liquefying enzyme, i.e., an a amylase (4 AAU per gram ds corn), and phytase (4 FTU per gram ds corn) were added and a timer was started to begin the incubation or primary liquefaction step. The slurry was allowed to incubate for 30 minutes in the presence of the amylase with or without added phytase. The phytase used in this experiment was BP-17 (see, supra). Although the phytase was added at the same time as the α-amylase in this example it may be added prior to the amylase.

The treated slurry was then placed in a water bath maintained at 90° C. to begin the secondary liquefaction step (2° liquefaction). Samples were taken to test for viscosity (by Brookfield) and DE (by Schoorls) at 0, 30, 60 and 90 minutes. The results are shown in FIGS. 14 and 15.

B. With Jet Cooking (Split Enzyme Dose)

A slurry of whole ground corn (obtained from Badger State Ethanol, Monroe, Wis.) was made with water containing 30% v/v thin stillage to a final concentration of about 32% ds. Corn solids were prepared in a jacked kettle. The slurry was mixed well and the pH of the slurry was measured (pH 5.2). This slurry was mixed in a jacketed kettle and brought up to the pretreatment temperature of 70° C. Just prior to reaching 70° C., the liquefying enzyme, i.e., an S242Q variant α amylase (3 AAU per gram ds corn), was added and a timer was started to begin the incubation or primary liquefaction step. The slurry was allowed to incubate for 30 minutes in the presence of the α-amylase with or without added phytase (4 FTU per gram ds corn). Although the phytase was added at the same time as the α-amylase in this example it may be added prior to the amylase.

The incubated slurry was then passed through a jet cooker (225° F.; 107.2° C.) which was preheated to the desired temperature using steam and water. The slurry was sent through the jet at maximum speed (1.5 setting) about 4 liters/minute. Using the first three loops of the hold coil resulted in a hold time of just over 3 minutes. After all of the water was displaced and the desired temperature held steady, an aliquot of solubilized corn mash was collected and placed in a secondary bath (overhead stirring) at 85° C. to begin the secondary liquefaction step (2° liquefaction). A second dose of the S242Q (1 AAU/gm ds) was added and the liquefaction continued for an additional 90 minutes. Samples were taken to test for viscosity (by Brookfield) and DE (by Schoorls) at 0, 30, 60 and 90 minutes. This liquefact was used in Example 10B.

C. Jet Cooking, Conventional

A slurry of whole ground corn (obtained from Badger State Ethanol, Monroe, Wis.) was made with water containing 30% v/v thin stillage to a final concentration of about 32% ds. Corn solids were prepared in a jacked kettle. The slurry was mixed well and the pH of the slurry was measured (pH 5.2) and adjusted to pH 5.8 with dilute NaOH. This slurry was mixed in a jacketed kettle and brought up to the pretreatment temperature of 70° C. Just prior to reaching 70° C., the liquefying enzyme, i.e., an S242Q variant a amylase (3 AAU per gram ds corn), was added and a timer was started to begin the incubation or primary liquefaction step. The slurry was allowed to incubate for 30 minutes in the presence of the α-amylase without added phytase.

The incubated slurry was then passed through a jet cooker (225° F.; 107.2° C.) which was preheated to the desired temperature using steam and water. The slurry was sent through the jet at maximum speed (1.5 setting) about 4 liters/minute. Using the first three loops of the hold coil resulted in a hold time of just over 3 minutes. After all of the water was displaced and the desired temperature held steady, an aliquot of solubilized corn mash was collected and placed in a secondary bath (overhead stirring) at 85° C. to begin the secondary liquefaction step (2° liquefaction). A second dose of the S242Q variant α-amylase (1 AAU/gm ds) was added and the liquefaction continued for an additional 90 minutes. Samples were taken to test for viscosity (by Brookfield) and DE (by Schoorls) at 0, 30, 60 and 90 minutes. The above experiment at a slurry pH of 5.5. See FIG. 22. This liquefact was used in Example 10A.

D. Results with and without Jet Cooking

Addition of BP-17 phytase during incubation (primary liquefaction) reduced the phytic acid content of the whole ground corn from 0.60% ds corn to 0.09% ds corn (>85% reduction) (FIG. 21). It is also very clear from FIGS. 14 and 15 that the a amylase were inactivated at a jet cooking temperature of 225° F. (107° C.) based on DE development or viscosity reduction. However, the inclusion of phytase prior to jet cooking (which it is believed to remove the phytic acid inhibition) resulted in a significant increase in the thermostability of the α amylases as shown by DE progression and viscosity reduction at 90° C. during the secondary liquefaction step. Similar results were seen with jet cooking (data not shown) as shown in FIGS. 14 and 15.

Example 9 Effect of BP-17 Phytase Concentration on α Amylase Stability at Low pH

The increase in the thermostability of α amylase due to the removal of the phytic acid inhibition of α amylase was further studied. The phytic acid was hydrolyzed using phytase prior to the secondary liquefaction of whole ground corn and the improvement in the pH stability at low pH was determined.

In a typical experiment, whole ground corn was slurried to a 32% (ds corn) by using a 70:30 ratio of water and thin stillage. The slurry pH was measured and found to be pH 5.2. The slurry was heated to 70° C. using water and steam in a jacketed kettle. The liquefaction enzyme, i.e., the S242Q variant α-amylase (4 AAU/gm ds corn), and varying concentrations of BP-17 (0-12 FTU/gm ds corn) were added and the slurry was pretreated by holding the temperature at 70° C. for 45 minutes. After 45 minutes of pretreatment, the slurry was placed in a 90° C. water bath. The liquefact was continuously stirred and held at 90° C. for 90 minutes. Samples were collected at 0, 30, 60 and 90 minutes. All samples were tested for DE (using the Schoorls method), and for viscosity (Brookfield viscometer spindle 2 at 20 rpm). The DE progression and viscosity data are summarized in FIGS. 16 and 17.

The results in FIGS. 16 and 17 show that the reduction of phytic acid inhibition of the S242Q variant α-amylase resulted in a significant increase in the low pH stability for activity as evidenced by a steady increase in the DE progression at 90° C. with a concomitant decrease in the viscosity of the liquefact. The data clearly showed that the S242Q variant α amylase can be successfully used in the liquefaction process for whole ground corn at a pH 5.2 if the inhibition of the phytic acid is eliminated. In FIG. 21, it can be seen that the rate of DE progression increases with the increased removal of phytic acid and reaches a maximum at 4 FTU/gm ds indicating that the phytase increases the thermostability of the S242Q variant α-amylase by removing phytic acid from the slurry.

Example 10 Effect of pH

In this example, the effect of pH on the S242Q variant α-amylase was examined.

In a typical experiment, whole ground corn was slurried to a 32% (ds corn) by using a 70:30 ratio of water and thin stillage. The slurry pH was measured and found to be pH 5.2. The pH was lowered to between 4.2 and 4.8 using H₂SO₄. The slurry was heated to 70° C. using water and steam in a jacketed kettle. The liquefaction enzyme, i.e., the S242Q variant (4 AAU/gm ds), and BP-17(4 FTU/gm ds) were added and the slurry was pretreated by holding the temperature at 70° C. for 45 minutes. After 45 minutes of pretreatment, the slurry was placed in a 90° C. water bath. The liquefact was continuously stirred and held at 90° C. for 90 minutes. Samples were collected at 0, 30, 60 and 90 minutes. All samples were tested for DE (using the Schoorls method), and for viscosity (Brookfield viscometer spindle 2 at 20 rpms). The DE progression and viscosity data are summarized in FIGS. 18 and 19.

The results showed that DE progression decreased with decreasing pH from 5.2 to 4.5. The enzyme was completely inactivated at pH 4.2.

Example 11 Effect on Ethanol Production

Liquefacts were used as fermentation feedstocks in ethanol fermentation for alcohol production. A slurry of whole ground corn (obtained from Badger State Ethanol, Monroe, Wis.) was mixed with water containing 30% v/v thin stillage to a final concentration of about 32% ds.

A. Conventional Process

The liquefact from Example 8C was used (Liquefact A).

The pH of the secondary liquefact was adjusted to 4.2 using H₂SO₄ prior to the simultaneous saccharification and fermentation (SSF) stage.

B. Low pH, Jet Cooking (Split Dose)

The liquefact from Example 8B was used (Liquefact B). No pH adjustment was done prior to SSF.

C. Simultaneous Saccharification and Sermentation

In each experiment tare weights of the vessels were obtained prior to preparation of media. A 32% DS corn ds liquefact (2 liters) was taken in a 2 L flask. Red Star Ethanol Red yeast (RED STAR (Lesaffre) inoculums were prepared by adding 10 grams of yeast and 1 gram of glucose to 40 grams of water under mild agitation for one hour. Five mls of each inoculum was added to equilibrated fermentors followed by the addition of G Zyme™ 480 Ethanol (Danisco US Inc, Genencor Division) at 0.4 GAU/gds.corn to initiate the simultaneous saccharification and fermentation. The initial gross weight was noted and the flask was placed in a water bath maintained at 32° C. The samples were taken at different intervals of time and analyzed for carbohydrate and ethanol content using HPLC. Fermentations were also carried out using one kilogram of each liquefact and weight loss during fermentation was measured at different intervals of time. Based on the weight loss due to loss of carbon dioxide, the alcohol was measured. At the conclusion of the fermentation, a final gross weight was obtained. The broth was quantitatively transferred into a 5 L round bottom vessel. Distillation was performed under vacuum until approximately 800 mls of ethanol was collected in a receptacle containing 200 mls water. The ethanol was diluted to 2 L and was analyzed by HPLC. The weight and DS of the still bottoms was obtained prior to drying. Residual starch analysis was performed on the DDGS. Stoichiometric calculations were performed based on weight loss, distillation, and residual starch analysis.

Ethanol calculation using CO₂ weight loss:

Ethanol production (mmol)=CO₂ loss (g)/88 Ethanol production (g)=(CO₂ loss (g)/88)*92=>CO₂ loss (g)* 1.045 Ethanol production (ml)=((CO₂ loss (g)/88)*92)/0.789=>CO₂ loss (g)×1.325

The data in FIG. 20 shows major difference in free sulphate and phytic acid content between the conventional process and the no pH adjustment process according to the invention. Removal of phytic acid inhibition of thermostable α amylase in the incubation resulted in the DDGS with reduced phytic acid content, higher free available phosphate and reduced sulfate. Thus, the process with no pH adjustment confers pH stability at low pH for liquefying thermostable α amylases in the starch liquefaction.

Example 12 Additional Methods

The following assays were used in the Examples described below. Any deviations from the protocols provided below are indicated in the Examples. In these experiments, a spectrophotometer was used to measure the absorbance of the products formed after the completion of the reactions.

A. Protein Content Determination

BCA (Bicinchoninic Acid) Assay

In these assays, BCA (Pierce) assay was used to determine the protein concentration in samples on microtiter plate (MTP) scale. In this assay system, the chemical and reagent solutions used were: BCA protein assay reagent, and Pierce dilution buffer (50 mM MES, pH 6.5, 2 mM CaCl₂, 0.005% TWEEN®-80). The equipment used was a SpectraMAX (type 340; Molecular Devices) MTP reader. The MTPs were obtained from Costar (type 9017).

In the test, 200 μl BCA Reagent was pipetted into each well, followed by 20 μl diluted protein. After thorough mixing, the MTPs were incubated for 30 minutes at 37° C. Air bubbles were removed, and the optical density (OD) of the solution within the wells was read at 562 nm. To determine the protein concentration, the background reading was subtracted form the sample readings. The OD₅₆₂ was plotted for protein standards (purified enzyme), to produce a standard curve. The protein concentration of the samples were interpolated from the standard curve.

Bradford Assay

In these assays, the Bradford dye reagent (Quick Start) assay was used to determine the protein concentration in samples on MTP scale. In this assay system, the chemical and reagent solutions used were: Quick Start Bradford Dye Reagent (BIO-RAD Catalog No. 500-0205), Dilution buffer (10 mM NaCl, 0.1 mM CaCl₂, 0.005% TWEEN®-80). The equipment used was a Biomek FX Robot (Beckman) and a SpectraMAX (type 340) MTP reader. The MTPs were from Costar (type 9017).

In the test, 200 μl Bradford dye reagent was pipetted into each well, followed by 15 μl dilution buffer. Finally 10 μl of filtered culture broth were added to the wells. After thorough mixing, the MTPs were incubated for at least 10 minutes at room temperature. Air bubbles were blown away and the ODs of the wells were read at 595 nm. To determine the protein concentration, the background reading (i.e., from un-inoculated wells) was subtracted form the sample readings. The obtained OD₅₉₅ values provide a relative measure of the protein content in the samples.

B. Microswatch Assay for Testing Enzyme Performance

The detergents used in this assay did not contain enzymes or the enzymes present in commercial detergents had been destroyed through heat deactivation as described elsewhere in this document. The equipment used included an Eppendorf Thermomixer and a SpectraMAX (type 340) MTP reader. The MTPs were obtained from Costar (type 9017).

Detergent Preparation (AATCC HDL; US Conditions)

Milli-Q water was adjusted to 6 gpg water hardness (Ca/Mg=3/1), and 1.5 g/l AATCC 2003 standard reference liquid detergent without brightener was added. The detergent solution was vigorously stirred for at least 15 minutes. Then, 5 mM HEPES (free acid) was added and the pH adjusted to 8.0.

Rice Starch Microswatch Assay for Testing Amylase Performance

Test detergents were prepared as described elsewhere in this document. The equipment used included a New Brunswick Innova 4230 shaker/incubator and a SpectraMAX (type 340) MTP reader. The MTPs were obtained from Corning (type 3641). Aged rice starch with orange pigment swatches (CS-28) were obtained from Center for Test Materials (Vlaardingen, Netherlands). Before cutting 0.25-inch circular microswatches, the fabric was washed with water. Two microswatches were placed in each well of a 96-well microtiter plate. The test detergent was equilibrated at 20° C. (North America) or 40° C. (Western Europe). 190 μl of detergent solution was added to each well of the MTP, containing microswatches. To this mixture, 10 μl of the diluted enzyme solution was added. The MTP was sealed with adhesive foil and placed in the incubator for 1 hour with agitation at 750 rpm at the desired test temperature (typically 20° C. or 40° C.). Following incubation, 150 μl of the solution from each well was transferred into a fresh MTP. This MTP was read at 488 nm using a SpectraMax MTP reader to quantify cleaning. Blank controls, as well as controls containing microswatches and detergent but no enzyme were also included.

Calculation of Enzyme Performance

The obtained absorbance value was corrected for the blank value (i.e., obtained after incubation of microswatches in the absence of enzyme). The resulting absorbance was a measure for the hydrolytic activity.

C. Amylase Concentration Determination by Antibody Titration

As described herein, α-amylase concentration and specific activity was determined by titration with an inhibitory polyclonal antibody. Polyclonal antibodies raised to Bacillus stearothermophilus α-amylase (AmyS) were found to be strongly inhibitory of AmyS and the α-amylase from Bacillus sp. TS23 (e.g., the binding is tight enough to produce a linear titration of activity loss). Therefore, this antibody can be used to measure enzyme concentration, which in turn is used to calculate specific activity. Briefly, the amount of enzyme inhibition produced by several known concentrations of antibody is measured. From this information, the concentration of antibody required for complete inhibition is extrapolated, which is equivalent to the enzyme concentration in the sample. A-amylase activity and inhibition was measured using the fluorogenic BODIPY-starch assay. The buffer was 50 mM MOPS, pH 7.0, containing 0.005% Tween-80.

A polyclonal antibody directed against purified AmyS was raised in a rabbit and purified by standard methods. An empirical “apparent concentration” value of an antibody stock solution was determined by measuring the inhibition of a sample of AmyS of known specific activity. Then the antibody sample was used to determine the concentration and specific activity of AmyS and TS23t variants. These values were used to create normalized 96-well enzyme stock plates, where all of the variants were diluted to a common concentration.

D. Native Protein Gel Electrophoresis

Electrophoretic mobility of variant protein samples was measured using the PhastGel system (GE Healthcare) on pre-cast native polyacrylamide gels (PhastGel Homogeneous) at either 7.5% or 12.5% concentration. Buffer strips (PhastGel Native) were used and consisted of pH 8.8 in 0.88 M L-Alanine, 0.25 M Tris buffer. Typical run conditions consisted of 400 V for 12.75 minutes with an anode-to-cathode distance of 3.7 cm.

Alternatively, electrophoretic mobility of variant protein samples was measured on 1 mm thick 0.5-1.5% agarose gels at various pH values (i.e. 5.8, 8.0 and 10.0) through a choice of a suitable buffer system. The electrophoresis is carried out under non-denaturing conditions. The Cathode-Anode length was 13.9 cm. A sample of 1-2 μg protein was mixed with 5% glycerol+0.05% bromophenol blue and loaded on each lane. Gels were run typically for 1 hour at 100V.

In either case gels were stained with Louiseville blue dye dissolved in 10% acetic acid and destained with 10% methanol and 10% acidic acid in water. It is possible to load between 12 and 20 protein variants simultaneously depending on native gel system used. As a consequence the electrophoretic mobility of a protein variant can be immediately assessed relative to charge ladder standards loaded on the same gel.

E. Detergent Heat Inactivation

Heat inactivation of commercial detergent formulas serves to destroy the enzymatic activity of any protein components while retaining the properties of non-enzymatic components. Thus this method was suitable for preparing commercially purchased detergents for use in testing the enzyme variants of the present invention. For North American (NA) and Western European (WE) heavy duty liquid laundry (HDL) detergents, heat inactivation was performed by placing pre-weighed liquid detergent (in a glass bottle) in a water bath at 95° C. for 2 hours. The incubation time for heat inactivation of North American (NA) and Japanese (JPN) heavy duty granular laundry (HDG) detergent was 8 hours and that for Western European (WE) HDG detergent was 5 hours. The incubation time for heat inactivation of NA and WE auto dish washing (ADW) detergents was 8 hours. The detergents were purchased from local supermarket stores. Both un-heated and heated detergents were assayed within 5 minutes of dissolving the detergent to accurately determine percentage deactivated. Enzyme activity was tested by the suc-AAPF-pNA assay.

For testing of enzyme activity in heat-inactivated detergents, working solutions of detergents were made from the heat inactivated stocks. Appropriate amounts of water hardness (6 gpg or 12 gpg) and buffer were added to the detergent solutions to match the desired conditions (Table 12-1). The solutions were mixed by vortexing or inverting the bottles.

TABLE 12-1 Laundry and Dish Washing Conditions Region Form Dose Detergent* Buffer Gpg pH T (° C.) Laundry (heavy duty liquid and granular) NA HDL 0.78 g/l P&G TIDE ® 2X 5 mM HEPES 6 8.0 20 WE HDL 5.0 g/L Henkel Persil 5 mM HEPES 12 8.2 40 WE HDG 8.0 g/L P&G Ariel 2 mM Na₂CO₃ 12 10.5 40 JPN HDG 0.7 g/L P&G TIDE ® 2 mM Na₂CO₃ 6 10.0 20 NA HDG 1.0 g/L P&G TIDE ® 2 mM Na₂CO₃ 6 10.0 20 Automatic Dish Washing WE ADW 3.0 g/L RB Calgonit 2 mM Na₂CO₃ 21 10.0 40 NA ADW 3.0 g/L P&G Cascade 2 mM Na₂CO₃ 9 10.0 40 *Abbreviations: Procter & Gamble (P&G); and Reckitt Benckiser (RB).

F. Terg-O-Tometer Assay for Cleaning Performance Determination

A standard protocol for assessing protein and carbohydrate soil cleaning was used whereby the soil level on a fabric swatch was measured before and after cleaning under standard conditions. The fabric swatches consisted of woven cotton fabric soiled with either maize starch, rice starch or a blood, milk and carbon black mixture, and were purchased from Testfabrics, Inc. (West Pittiston, Pa.). Maize Starch (EMPA 161) and Blood, Milk, Carbon Black (EMPA 116) technical soils were produced by EMPA Test materials AG (St. Gallen, Switzerland). Rice Starch (CFT CS-28) soils were produced by the Center for Testmaterials BV (Vlaardingen, Netherlands). Each stain was measured before and after treatment by optical reflectance using a Minolta Reflectometer CR-410 set to a D65 (6500° K) standard illuminant. The difference in the L, a, b values was converted to total color difference (dE), as defined by the CIE-LAB color space. Cleaning of the stains are expressed as percent stain removal index (% SRI) by taking a ratio between the color difference before and after washing and comparing it to the difference of unwashed soils (before wash) to unsoiled fabric.

Cleaning experiments were conducted in a Terg-o-tometer (United States Testing Co., Hoboken, N.J.) equipped with 6 stainless steel 2 L pots fitted with overhead agitators. Each treatment was conducted in 1 L total volume consisting of either 6 grains per gallon 3:1 (calcium:magnesium) water hardness or 12 grains per gallon water hardness. Detergents used in the wash experiments were 1.5 g/L AATCC HDL WOB 2003 liquid detergent with 5 mM HEPES buffer at pH 8, 0.7 g/L AATCC HDD WOB 1993 granular detergent, 8 g/L IEC A*60456 granular detergent with perborate and TAED bleach, or 5 g/L Persil Power Gel liquid detergent. Enzyme was added directly into the wash solution and reactions were then initiated by addition of either 40 g/L or 200 g/L of soiled and ballast fabric. The washing reactions were agitated at 100 rpm for 10, 15, or 40 minutes at 20° C., 25° C., 30° C., 40° C., or 50° C. Following cleaning, swatches were rinsed for 3 minutes in tap water, spun in a front-loading washing machine at 1000 rpm to remove excess water, and dried in a dryer at low heat on a permanent press cycle for approximately 45 minutes. Comparison of the extent of soil removal was assessed by reflectometry and expressed as the percent soil removal index (% SRI). The control condition did not contain enzyme and the positive control consisted of various doses of benchmark commercial enzymes.

G. Bodipy-Starch Assay for Determination of Amylase Activity

The Bodipy-starch assay was performed using the ENZCHEK® Ultra Amylase Assay Kit (E33651, Invitrogen). A 1 mg/mL stock solution of the DQ starch substrate was prepared by dissolving the contents of the vial containing the lyophilized substrate in 100 μL of 50 mM sodium acetate buffer at pH 4.0. The vial was vortexed for about 20 seconds and left at room temperature, in the dark, with occasional mixing until dissolved. 900 μL of assay buffer (50 mM sodium acetate with 2.6 mM CaCl₂ pH 5.8) was added and the vial vortexed for about 20 seconds. The substrate solution was stored at room temperature, in the dark, until ready to use or at 4° C. For the assay, a 100 μg/mL of working solution of the DQ substrate was prepared from the 1 mg/mL substrate solution in the assay buffer. 190 μL of 100 μg/mL substrate solution was added to each well in a 96-well flat-bottom microtiter plate. 10 μL of the enzyme samples were added to the wells, mix for 30 seconds using a thermomixer at 800 rpms. A blank sample that contains buffer and substrate only (no-enzyme blank) was included in the assay. The rate of change of fluorescence intensity was measured (excitation: 485 nm, emission: 520 nm) in a fluorescence microtiter plate reader at 25° C. for 5 minutes.

H. Corn Flour Hydrolysis for Determination of Amylase Activity

Starch Hydrolysis of Corn Flour Substrate Assay for Enymatic Activity. Organic corn flour (Azure Farms, lot no. 03227) was evenly spread into Greiner 96-well microplate, polypropylene, black, flat bottom chimney wells, (Cat. No. 655209), using a solids dispensing device (V&P Scientific). 85 μL of 20 mM sodium acetate pH 5.6 were added to each well and mixed. A foil seal was applied to the top of the plate and the plate pre-incubated at 70° C. in the Thermomixer for 20-30 minutes. Enzyme samples were diluted in Agilent polypropylene plate (5042-1385) in 20 mM sodium acetate buffer. 11 μL of diluted enzyme samples were added to the substrate plate and the plate sealed firmly with another foil. Plates were then transferred to Labnet VorTemp 56 Incubator/Shaker with metal blocks, (Cat. No. 52056A) pre-heated to 95° C. and the shake speed set to 500 rpm. The incubation was continued for 30 minutes. At the end of the incubation, the plates were rapidly cooled in an ice bucket and the starch hydrolysis reaction was stopped by addition of 100 μL of 0.1 N H₂SO₄ to each well. The plate was mixed briefly and the starch hydrolysis reaction products were either analyzed by the PAHBAH assay or HPLC.

Colorimetric detection of Soluble Sugar Concentrations from Enzymatic Hydrolysis of Corn Flour Substrate. Aliquots of 80 μL of 0.5 N NaOH were added to all wells of an empty PCR plate followed by 20 μL of PAHBAH reagent (5% w/v p-hydroxybenzoic acid hydrazide (PAHBAH, Sigma # H9882, dissolved in 0.5 N HCl) and mixed (PAHBAH reaction plate). 10 μL of the starch hydrolysis reaction supernatants were added to the PAHBAH reaction plate. All plates were sealed and placed in the thermocycler (MJ Research Tetrad), programmed for 2 minutes at 95° C., and then cooled to 20° C. Samples of 80 μL of the developed PAHBAH reaction mixtures were transferred to a read plate and absorbance was measured at 405 nm in a spectrophotometer.

HPLC Determination of Soluble Sugar Concentrations from Enzymatic Hydrolysis of Corn Flour Substrate. Soluble sugar standards (DP1-DP7) obtained from Sigma (St. Louis, Mo.) were all diluted in Milli-Q water to 100 mg/mL and used for converting peak area for the sugars to actual sugar concentrations. The quenched plate from the starch hydrolysis assay was spun in a Beckman Coulter Allegra 6R Centrifuge for 5 minutes at 3000 rpm 25° C. The supernatant was pipetted from the spun plate and transferred to a Multiscreen-HV filter plate (Catalog No. MAHVN4550). The filter plate was spun over an Agilent HPLC plate in the Hettich Rotanta centrifuge for 10 minutes at 6,000 rpm 25° C. 50 μL of 0.01 N sulfuric acid mobile phase (0.1 N sulfuric acid diluted 10× with Milli-Q water) was transferred to each well of another clean Agilent HPLC plate. The filtered plate was briefly mixed and 50 μL of the filtrate was transferred the corresponding wells in the plate with 50 μL per well of mobile phase. Diluted sugar standards were added to empty wells in the plate to be included in the calibration. The contents were mixed briefly on a platform shaker and the plate covered with a Nalgene Pre-slit Well Cap. The HPLC column (Bio-Rad Aminex HPX-87H column Cat No. 125-0140) was prepared ahead of time with 2 L of mobile phase running at a constant flow rate of 0.6 mL/minute. All samples in the plate were run with 20 μL injection volume and analyzed using AMINEXH.M and RID (refractive index) as the detector. After the run was completed, the flow rate in the HPLC was dropped down to 0.05 mL/min.

I. Determination of Starch Viscosity Reduction by Amylase

In this assay, viscosity reduction of corn starch substrate solution was measured in a viscometer. The corn starch substrate slurry was made up fresh in batch mode with 30% corn flour dry solids in distilled water and adjusted to pH 5.8 using sulfuric acid. For each run, 50 grams of the slurry (15 grams dry solids) was weighed out and pre-incubated for 10 minutes to warm up to 70° C. Upon amylase addition, the temperature was immediately ramped up from 70° C. to 85° C. with a rotation speed of 75 rpm. Once the temperature of the slurry and amylase mixture reached 85° C., the temperature was held constant and viscosity was monitored for an additional 30 minutes.

J. Measurement of Enzyme Binding to Macromolecular Substrates

Binding assays were done to determine substrate binding of Amylase (AmyS) charge ladder variants (charge change=−12 to +12 relative to wild-type AmyS) to corn stover and bagasse. Substrates used included bagasse (sugarcane bagasse from Brazil, dilute-acid pre-treated by National Renewable Energy Laboratory, washed and buffered at pH 5), AFEX (ammonia fiber expansion corn stover), and PCS (dilute sulfuric acid pre-treated corn stover, washed and adjusted to pH 5). All substrates were brought to the desired percentage solids prior to use.

Amylase Binding: Amylase charge ladder variants were purified and diluted to 200 ppm for testing. A 1% cellulose bagasse solution was prepared in borate buffer (40 mM, pH8.5, 0.016% Tween80). 150 μl of the bagasse solution was added into each well in a microtiter filtration plate. 150 μl of borate buffer was added into a set of separate wells, which served as controls. 10 μl of amylase charge ladder variants was added into the filtration plate, each condition was in duplicates. The plate was incubated at room temperature for 2 hours. The filtrate was collected and amylase activity in the supernatant was measured by BODIPY-starch assay.

Measurement of Enzyme Binding to Microswatches: Amylase variants were incubated with or without CS-28 rice starch micro swatches under standard wash conditions for 30 min. The amount of free enzyme was measured by the BODIPY-starch assay. The fraction of enzyme bound to the microswatches was calculated as follows: Fraction bound=(Activity of enzyme in absence of swatch—Activity of enzyme in presence of swatch)/(Activity of enzyme in absence of swatch).

K. Geobacillus Stearothermophilus Amylase Protein Quantitation

The G. stearothermophilus amylase protein was quantitated by competitive immunoassay. Briefly, purified G. stearothermophilus amylase was labeled with a fluorescent dye (fluorescein) and antibody to G. stearothermophilus amylase was labeled with a quencher dye (tetramethylrhodamine). The fluorescence signal of the fluorescein-amylase conjugate is quenched upon binding of the quencher-labeled antibody. The presence of free amylase in the sample competes for the quencher-labeled antibody, resulting in an increase of the fluorescence signal. Therefore, the strength of the fluorescence signal is proportional to the amount of free amylase in the sample. The assay was calibrated with purified G. stearothermophilus amylase of known concentrations.

Labeling of G. stearothermophilus Amylase with Fluorescent Dye: Purified Geobacillus stearothermophilus amylase was labeled with fluorescein isothiocyanate (Molecular Probes, Eugene Oreg.) at pH 9.5 in 50 mM sodium carbonate buffer according to the manufacturer's protocol. At the end of the reaction, the protein was separated from unbound dye by gel filtration over Sephadex G-25 (Sigma, St Louis, Mo., USA) in phosphate-buffered saline.

Antibody Preparation and Labeling with Quencher Dye: Antibody to Geobacillus stearothermophilus amylase was prepared by immunization of rabbits and recovery of the antiserum. The antiserum was stored at −20° C. until use. Preparation of the immunoglobulin fraction by carried out by ammonium sulfate precipitation; 15 ml. 3.75 M ammonium sulfate was added to 20 ml antiserum and allowed to sit at 4° C. for 60 minutes before centrifugation at 2,000 g to recover the precipitate. The recovered precipitate was washed twice by resuspension and sedimentation in 10 ml ice-cold 1.6 M ammonium sulfate. The final precipitate was dissolved in 4 ml. water and dialyzed against 50 mM sodium carbonate pH 9.5 at 4° C. The protein concentration was estimated to be 29.2 mg/ml. by A₂₈₀ using the extinction coefficient ∈_(1%)=15. The immunoglobulin fraction was labeled with tetramethyl rhodamine isothiocyanate (Sigma, St. Louis, Mo.) in 50 mM sodium carbonate pH 9.5. Unbound dye was then removed by gel filtration on a column of Sephadex G-25 equilibrated with phosphate-buffered saline containing 0.1% deoxycholate.

Assay Procedure: The immunoassay was carried out in 96-well microtiter plates (Corning #3650). The concentration of the fluorescein-amylase was adjusted such that the final concentration in the assay would be the middle of the desired standard curve. Similarly, the concentration of the quencher-antibody was adjusted such that the final concentration would allow for maximum modulation of the fluorescence signal. Using an automated liquid handling system, 5 μL each of sample, fluorescein-amylase, and quencher antibody were added to 180 μL phosphate-buffered saline containing 2% (w/v) polyethylene glycol 8000 (Sigma, St. Louis, Mo., USA). After briefly shaking, the plates were allowed to incubate at room temperature for one hour the fluorescence signal was determined using a fluorescence plate reader (Molecular Devices) with excitation and emission filters set to 495 nm and 520 nm, respectively.

Example 13 Amylase Production in B. subtilis

In this Example, production of a mutant truncated form G. stearothermophilus amylase α amylase (having a S242Q mutation and a 29 amino acid deletion from the C-terminus; also referred to herein as S242Q) and variants thereof in B. subtilis are described. Transformation was performed as known in the art (see, e.g., WO 02/14490). Briefly, the gene encoding the parent amylases was cloned into the pHPLT expression vector, which contains the LAT promoter (PLAT), a sequence encoding the LAT signal peptide (preLAT), followed by PstI and HpaI restriction sites for cloning.

The coding region for the LAT signal peptide is shown below:

(SEQ ID NO: 20) atgaaacaac aaaaacggct ttacgcccga ttgctgacgc tgttatttgc gctcatcttc ttgctgcctc attctgcagc ttcagca.

The amino acid sequence of the LAT signal peptide is shown below:

MKQQKRLYAR LLTLLFALIF LLPHSAASA (SEQ ID NO: 21)

The amino acid sequence of the mature truncated S242Q amylase with the substituted amino acid shown in italics was used as the basis for making the variant libraries described herein:

(SEQ ID NO: 22) AAPFNGTMMQ YFEWYLPDDG TLWTKVANEA NNLSSLGITA LWLPPAYKGT SRSDVGYGVY DLYDLGEFNQ KGTVRTKYGT KAQYLQAIQA AHAAGMQVYA DVVFDHKGGA DGTEWVDAVE VNPSDRNQEI SGTYQIQAWT KFDFPGRGNT YSSFKWRWYH FDGVDWDESR KLSRIYKFRG IGKAWDWEVD TENGNYDYLM YADLDMDHPE VVTELKNWGK WYVNTTNIDG FRLDAVKHIK FQFFPDWLSY VRSQTGKPLF TVGEYWSYDI NKLHNYITKT NGTMSLFDAP LHNKFYTASK SGGAFDMRTL MTNTLMKDQP TLAVTFVDNH DTEPGQALQS WVDPWFKPLA YAFILTRQEG YPCVFYGDYY GIPQYNIPSL KSKIDPLLIA RRDYAYGTQH DYLDHSDIIG WTREGVTEKP GSGLAALITD GPGGSKWMYV GKQHAGKVFY DLTGNRSDTV TINSDGWGEF KVNGGSVSVW VPRKTT.

The coding region for the mature AmyS amylase is shown below:

(SEQ ID NO: 23) gccgcaccgt ttaacggtac catgatgcag tattttgaat ggtacttgcc ggatgatggc acgttatgga ccaaagtggc caatgaagcc aacaacttat ccagccttgg catcaccgct ctttggctgc cgcccgctta caaaggaaca agccgcagcg acgtagggta cggagtatac gacttgtatg acctcggcga attcaatcaa aaagggaccg tccgcacaaa atatggaaca aaagctcaat atcttcaagc cattcaagcc gcccacgccg ctggaatgca agtgtacgcc gatgtcgtgt tcgaccataa aggcggcgct gacggcacgg aatgggtgga cgccgtcgaa gtcaatccgt ccgaccgcaa ccaagaaatc tcgggcacct atcaaatcca agcatggacg aaatttgatt ttcccgggcg gggcaacacc tactccagct ttaagtggcg ctggtaccat tttgacggcg ttgactggga cgaaagccga aaattaagcc gcatttacaa attccgcggc atcggcaaag cgtgggattg ggaagtagac acggaaaacg gaaactatga ctacttaatg tatgccgacc ttgatatgga tcatcccgaa gtcgtgaccg agctgaaaaa ctgggggaaa tggtatgtca acacaacgaa cattgatggg ttccggcttg atgccgtcaa gcatattaag ttcagttttt ttcctgattg gttgtcgtat gtgcgttctc agactggcaa gccgctattt accgtcgggg aatattggag ctatgacatc aacaagttgc acaattacat tacgaaaaca aacggaacga tgtctttgtt tgatgccccg ttacacaaca aattttatac cgcttccaaa tcagggggcg catttgatat gcgcacgtta atgaccaata ctctcatgaa agatcaaccg acattggccg tcaccttcgt tgataatcat gacaccgaac ccggccaagc gctgcagtca tgggtcgacc catggttcaa accgttggct tacgccttta ttctaactcg gcaggaagga tacccgtgcg tcttttatgg tgactattat ggcattccac aatataacat tccttcgctg aaaagcaaaa tcgatccgct cctcatcgcg cgcagggatt atgcttacgg aacgcaacat gattatcttg atcactccga catcatcggg tggacaaggg aaggggtcac tgaaaaacca ggatccgggc tggccgcact gatcaccgat gggccgggag gaagcaaatg gatgtacgtt ggcaaacaac acgctggaaa agtgttctat gaccttaccg gcaaccggag tgacaccgtc accatcaaca gtgatggatg gggggaattc aaagtcaatg gcggttcggt ttcggtttgg gttcctagaa aaacgaccgt ttctaccatc gctcggccga tcacaacccg accgtggact ggtgaattcg tccgttggac cgaaccacgg ttggtggcat ggcct

The amino acid sequence of the mature AmyS amylase was used as the basis for making the AmyS variant libraries, and is provided as SEQ ID NO:2.

The PCR products were purified using Qiaquik columns from Qiagen, and resuspended in 50 μL of deionized water. 50 μL of the purified DNA was digested with HpaI (Roche) and PstI (Roche) and the resultant DNA resuspended in 30 μL of deionized water. 10-20 ng/μL of the DNA was cloned into plasmid pHPLT using PstI and HpaI cloning sites. The ligation mixtures were directly transformed into competent B. subtilis cells (genotype: Δvpr, ΔwprA, Δmpr-ybfJ, ΔnprB). The B. subtilis cells have a competency gene (comK) which is placed under a xylose inducible promoter, so xylose was used to induce competency for DNA binding and uptake (see Hahn et al. (1996) Mol. Microbiol. 21:763-775).

The elements of plasmid pHPLT-AmyS include: pUB110=DNA fragment from plasmid pUB110 (McKenzie et al. (1986) Plasmid 15: 93-103). Plasmid features include: ori-pUB110=origin of replication from pUB110, neo=neomycin resistance gene from pUB110, Plat=transcriptional promoter from B. licheniformis amylase, Pre LAT=signal peptide from B. licheniformis amylase, SAMY 425ss=The coding region for truncated AmyS gene sequence (replaced by the coding regions for each truncated AmyS variant expressed in this study), Terminator=transcriptional terminator from B. licheniformis amylase.

Example 14 Expression of Enzyme Variants

This Example describes the methods used to express various recombinant enzymes of the transformed B. subtilis of the preceding Examples on a 2 ml scale.

B. subtilis clones containing AmyS (or a variant thereof) or S242Q (or a variant thereof) expression vectors were replicated with a steel 96-well replicator from glycerol stocks into 96-well culture plates (BD, 353075) containing 150 μl of LB media+10 μg/ml neomycin, grown overnight at 37° C., 220 rpm in a humidified enclosure. A 100 μl aliquot from the overnight culture was used to inoculate 2,000 μl defined media+10 μg/ml neomycin in 5 ml plastic culture tubes. The cultivation media was an enriched semi-defined media based on MOPS buffer, with urea as major nitrogen source, glucose as the main carbon source, and supplemented with 1% soytone and 5 mM calcium for robust cell growth. Culture tubes were incubated at 37° C., 250 rpm, for 72 hours. Following this incubation, the culture broths were centrifuged for 10 minutes at 3,000×g. The supernatant solution was decanted into 15 ml polypropylene conical tubes and 80 μL of each sample were aliquoted into 96 well plates for protein quantitation.

Example 15 Production of Enzyme Variants

This Example describes the production of enzyme charge ladders and combinatorial charge libraries.

Enzyme Charge Ladders

Multiple protein variants spanning a range of physical properties of interest are selected from existing libraries or are generated by site-directed mutagenesis techniques as known in the art (see, e.g., U.S. Pat. Pub. No. 2008-0293610). This defined set of probe proteins is then assayed in a test of interest.

Exemplary amylase charge ladder variants are shown in the following tables and assayed as described herein. In these tables, the charge change is relative to the parent enzyme.

The sequence of the AmyS gene was provided to Gene Oracle (Mountain View, Calif.) for the synthesis of the 28 charge ladder variants shown in Tables 15-1 and 15-2. Gene Oracle synthesized and cloned the AmyS variants into vector pGov4 and transformed them into E. coli. DNA isolated from minipreps, as well as an agar stab were supplied for each variant.

The variants were PCR amplified and cloned into the pHPLT B. subtilis expression vector. The variants were amplified as a PstI-HindIII fragment from plasmid pGov4 using primers:

Satori F (SEQ ID NO: 24) 5′-CTCATCTTCTTGCTGCCTCATTCTGCAGCTTC-3′; and Satori R (SEQ ID NO: 25) 5′-TTATCCTTTACCTTGTCTCCAAGC-3′.

The PCR products were purified using Qiagen Qiaquik columns, and resuspended in 50 μL of milliQ water. 50 μL of the purified DNA was digested with HindIII (Roche) and PstI (Roche) and the resultant DNA resuspended in 30 μL of deionized water. 10-20 ng/μL of the DNA was cloned into plasmid pHPLT using PstI and HpaI cloning sites. The ligation mixtures were directly transformed into competent B. subtilis cells (genotype: amyE::xylRPxylAcomK-phleo). These B. subtilis cells have a competency gene (comK) which is placed under a xylose inducible promoter, so xylose was used to induce competency for DNA binding and uptake.

TABLE 15-1 First AmyS Charge Ladder Number AmyS Variant Δ Charge 1-6 R308Q R483Q K171Q K383Q K447Q K471Q −12 N28D N224D N271D N281D Q86E Q89E 1-5 R308Q R483Q K171Q K383Q K447Q N28D −10 N224D N271D N281D Q86E 1-4 R308Q R483Q K171Q K383Q N28D N224D −8 N271D N281D 1-3 R308Q R483Q K171Q N28D N224D N271D −6 1-2 R308Q R483Q N28D N224D −4 1-1 R308Q N28D −2 AmyS Parent 0 2-1 D318N N28R +2 2-2 D318N D306N N28R N224R +4 2-3 D318N D306N D19N N28R N224R N271R +6 2-4 D318N D306N D19N D393N N28R N224R N271R +8 N281R 2-5 D318N D306N D19N D393N D458N N28R N224R +10 N271R N281R Q86R 2-6 D318N D306N D19N D393N D458N E29Q N28R +12 N224R N271R N281R Q86R Q89R

TABLE 15-2 Second AmyS Charge Ladder 3-7 Q97R Q319R Q358E Q443E N28D N224D N271D N281D −12 Q86E Q89E R308Q R483Q K171Q K383Q K447Q K471Q 3-6 Q97R Q319R Q358E Q443E N28D N224D N271D N281D −10 Q86E R308Q R483Q K171Q K383Q K447Q 3-5 Q97R Q319R Q358E Q443E N28D N224D N271D N281D −8 R308Q R483Q K171Q K383Q 3-4 Q97R Q319R Q358E Q443E N28D N224D N271D R308Q −6 R483Q K171Q 3-3 Q97R Q319R Q358E Q443E N28D N224D R308Q R483Q −4 3-2 Q97R Q319R Q358E Q443E N28D −2 3-1 Q97R Q319R Q358E Q443E 0 4-1 Q97R Q319R Q358E Q443E N28K D318N +2 4-2 Q97R Q319R Q358E Q443E N28K N224K D318N D306N +4 4-3 Q97R Q319R Q358E Q443E N28K N224K N271K D318N +6 D306N D19N 4-4 Q97R Q319R Q358E Q443E N28K N224K N271K N281K +8 D318N D306N D19N D393N 4-5 Q97R Q319R Q358E Q443E N28K N224K N271K N281K +10 Q86R D318N D306N D19N D393N D458N 4-6 Q97R Q319R Q358E Q443E N28K N224K N271K N281K +12 Q86R Q89R D318N D306N D19N D393N D458N E29Q 5-1 Q97R Q319R Q358E Q443E N28D R308Q S242E −3 5-2 Q97R Q319R Q358E Q443E N28D N224D R308Q S242E −4 5-3 Q97R Q319R Q358E Q443E N28D N224D R308Q S242Q −3

TABLE 15-3 AmyS-S242Q Charge Ladder AmyS-S242Q Variant Δ Charge Q97E-Q319E-Q358E-Q443E −4 Q97E-Q319E-Q358E −3 Q97E-Q319E −2 Q97E −1 Q97R-Q319E 0 Parent AmyS-S242Q 0 Q97R +1 Q97R-Q319R +2 Q97R-Q319R-Q358R +3 Q97R-Q319R-Q358R +4 Enzyme Combinatorial Charge Libraries (CCL): Generation of G. stearothermophilus AmyS-S242Q CCL

The AmyS-S242Q plasmid DNA was isolated from a transformed B. subtilis strain (gentotype: ΔaprE, ΔnprE, amyE::xylRPxylAcomK-phleo) and sent to DNA2.0 Inc. as the template for CCL construction. A request was made to DNA2.0 Inc. (Mountain View, Calif., USA) for the generation of positional libraries at each of the four sites in AmyS-S242Q (S242Q) amylase that are shown in Table 15-4. Variants were supplied as glycerol stocks in 96-well plates.

The AmyS S242Q combinatorial charge library was designed by identifying the following four residues: Gln-97, Gln 319, Gln 358, and Gln 443. A four site, 81-member CCL was created by making all combinations of three possibilities at each site: wild-type, arginine, or aspartic acid.

TABLE 15-4 S242Q CCL Variants Variant # Q97 Q319 Q358 Q443 Δ Charge 1 Q97E Q319E Q358E Q443E −4 2 Q97E Q319E Q358E Q443R −2 3 Q97E Q319E Q358E — −3 4 Q97E Q319E Q358R Q443E −2 5 Q97E Q319E Q358R Q443R 0 6 Q97E Q319E Q358R — −1 7 Q97E Q319E — Q443E −3 8 Q97E Q319E — Q443R −1 9 Q97E Q319E — — −2 10 Q97E Q319R Q358E Q443E −2 11 Q97E Q319R Q358E Q443R 0 12 Q97E Q319R Q358E — −1 13 Q97E Q319R Q358R Q443E 0 14 Q97E Q319R Q358R Q443R +2 15 Q97E Q319R Q358R — +1 16 Q97E Q319R — Q443E −1 17 Q97E Q319R — Q443R +1 18 Q97E Q319R — — 0 19 Q97E — Q358E Q443E −3 20 Q97E — Q358E Q443R −1 21 Q97E — Q358E — −2 22 Q97E — Q358R Q443E −1 23 Q97E — Q358R Q443R +1 24 Q97E — Q358R — 0 25 Q97E — — Q443E −2 26 Q97E — — Q443R 0 27 Q97E — — — −1 28 Q97R Q319E Q358E Q443E −2 29 Q97R Q319E Q358E Q443R 0 30 Q97R Q319E Q358E — −1 31 Q97R Q319E Q358R Q443E 0 32 Q97R Q319E Q358R Q443R +2 33 Q97R Q319E Q358R — +1 34 Q97R Q319E — Q443E −1 35 Q97R Q319E — Q443R +1 36 Q97R Q319E — — 0 37 Q97R Q319R Q358E Q443E 0 38 Q97R Q319R Q358E Q443R +2 39 Q97R Q319R Q358E — +1 40 Q97R Q319R Q358R Q443E +2 41 Q97R Q319R Q358R Q443R +4 42 Q97R Q319R Q358R — +3 43 Q97R Q319R — Q443E +1 44 Q97R Q319R — Q443R +3 45 Q97R Q319R — — +2 46 Q97R — Q358E Q443E −1 47 Q97R — Q358E Q443R +1 48 Q97R — Q358E — 0 49 Q97R — Q358R Q443E +1 50 Q97R — Q358R Q443R +3 51 Q97R — Q358R — +2 52 Q97R — — Q443E 0 53 Q97R — — Q443R +2 54 Q97R — — — +1 55 — Q319E Q358E Q443E −3 56 — Q319E Q358E Q443R −1 57 — Q319E Q358E — −2 58 — Q319E Q358R Q443E −1 59 — Q319E Q358R Q443R +1 60 — Q319E Q358R — 0 61 — Q319E — Q443E −2 62 — Q319E — Q443R 0 63 — Q319E — — −1 64 — Q319R Q358E Q443E −1 65 — Q319R Q358E Q443R +1 66 — Q319R Q358E — 0 67 — Q319R Q358R Q443E +1 68 — Q319R Q358R Q443R +3 69 — Q319R Q358R — +2 70 — Q319R — Q443E 0 71 — Q319R — Q443R +2 72 — Q319R — — +1 73 — — Q358E Q443E −2 74 — — Q358E Q443R 0 75 — — Q358E — −1 76 — — Q358R Q443E 0 77 — — Q358R Q443R +2 78 — — Q358R — +1 79 — — — Q443E −1 80 — — — Q443R +1 81 (parent) Q97 Q319 Q358 Q443 0

Example 16 Enzyme Wash Performance

This Example describes the testing of S242Q variant in a microswatch assay 1.0 μg/ml in AATCC HDL detergent or 5 mM HEPES buffer under varying ionic strength. The methods provided in Example 12 were used (see, e.g., “Rice Starch Microswatch Assay for testing Amylase Performance”).

There is an optimal net charge change for cleaning performance for enzyme in AATCC HDL detergent. Performance is measured in terms of relative cleaning performance observed in a rice starch microswatch activity assay. A value of around 1.0 indicates top cleaning performance in this assay. This is an example of optimizing a protein physical property (e.g., net charge) for improving a given outcome or benefit (e.g., cleaning performance in a liquid laundry detergent). The charge optimum identified with this limited set of probe proteins coincides with the optimum charge observed when measuring the entire charge combinatorial library. The use of probe proteins is therefore predictive of the behavior of the entire library.

According to the Debye-Hiickel theory (Israelachivili, Intermolecular and Surface Forces, Second Edition: With Applications to Colloidal and Biological Systems, Academic Press 2^(nd) Ed. [1992]), electrostatic interactions are governed primarily by the strength of double-layer forces between interacting species at constant potential or constant charge (enzymes, substrates, fabric, and detergent), their size, and the dielectric constant of the surrounding medium. In order to characterize the electrostatic behavior of particles in a complex medium, such as a detergent formulation, their interaction in a reduced environment possessing the same Debye screening length is sufficient. This was accomplished by choosing a buffer of matching pH and conductivity to that of the detergent under wash conditions. An appropriate buffer for such testing is 5 mM HEPES buffer at pH 8.0 with varying amounts of indifferent electrolyte, such as NaCl. Addition of 2.5 mM NaCl to this buffer matches the pH and conductivity of typical North American wash conditions. Addition of 100 mM NaCl is representative of Japanese and European wash conditions, typically higher in ionic strength due to both increased water hardness and detergent concentrations.

FIG. 23 shows that positive charge S242Q variants were superior for cleaning of rice starch microswatch under North American laundry conditions. Similarly, positive charge variants of another α-amylase (i.e., TS23t) were superior for cleaning of rice starch microswatch under North American laundry conditions (FIG. 24), demonstrating that charge mutations have a similar effect in different α-amylases. Positive charge S242Q variants also exhibit higher specific activity for granular corn starch substrates hydrolysis (FIG. 25).

Starch liquefaction by the AmyS charge ladder variants was determined by monitoring the final viscosity following liquefaction of corn starch. A low viscosity value is indicative of breakdown of starch polysaccharides. As shown in FIG. 14 a charge optimum (e.g., −4 to −2) was observed for liquefaction. AmyS variants that were too negative (e.g., −12 to −10) exhibited very high final viscosities, and variants that were too positive (e.g., +6 or greater) exhibited even higher final viscosities (e.g., beyond limits of lab instrumentation due to torque overload).

Example 17 Thermostability

This Example describes determining the relationship between protein charge and thermal stability. Amylase assays were based on BODIPY starch hydrolysis before and after heating the culture supernatant. The same chemical and reagent solutions were used as described in Example 12.

Thermal Stability Assay for α-Amylases

The filtered culture supernatants were serially diluted in 50 mM sodium acetate+2 mM CaCl₂ pH 5.8 with 0.002% Tween. 10 μl of each diluted culture supernatant was assayed to determine the initial amylase activity by the BODIPY starch assay. 50 μl of each diluted culture supernatant was placed in a VWR low profile PCR 96 well plate. 30 μL of mineral oil was added to each well as a sealant. The plate was incubated in a BioRad DNA engine Peltier Thermal Cycler at 95° C. for 30 or 60 minutes depending on the stability of the parent enzyme. Following incubation, the plate was cooled to 4° C. for min and then kept at room temperature. 10 μl of each sample was added to a fresh plate and assayed to determine the final amylase activity by the BODIPY starch assay as described in Example 1.

Calculation of Thermostability

The residual activity of a sample was expressed as the ratio of the final absorbance and the initial absorbance, both corrected for blanks. A higher index indicates a more thermally stable variant. This is an example of optimizing a protein physical property, in this case net charge, for improving enzyme thermal stability for a liquid laundry application.

Thermostability of the variants were assessed as described above. Thermostability winners from the S242Q CCL are listed in Table 17-1. Winners were defined as those having a ratio of mutant residual activity to parent (i.e., S242Q) residual activity greater than 1. FIG. 30 shows the residual activity of the AmyS first charge ladder as a function of charge change relative to wild type. The thermal stability employed in this assay is described in example 12. Once again as evidenced from the Figure accumulation of extreme negative charges (−12) or positive charges (+4) relative to the wild type enzyme are detrimental for thermal stability. This is an example of optimizing a protein physical property, in this case net charge, for improving enzyme thermal stability for a liquid laundry application.

TABLE 17-1 S242Q CCL - thermal stability winners Mut residual act./WT residual Variant # 97 319 358 443 act. 2 Q97E Q319E Q358E Q443R 1.12 10 Q97E Q319R Q358E Q443E 1.12 13 Q97E Q319R Q358R Q443E 1.36 14 Q97E Q319R Q358R Q443R 1.16 15 Q97E Q319R Q358R 1.37 17 Q97E Q319R Q443R 1.29 18 Q97E Q319R 1.11 27 Q97E 1.16 32 Q97R Q319E Q358R Q443R 1.18 37 Q97R Q319R Q358E Q443E 1.29 38 Q97R Q319R Q358E Q443R 1.22 39 Q97R Q319R Q358E 1.21 40 Q97R Q319R Q358R Q443E 1.20 41 Q97R Q319R Q358R Q443R 1.26 42 Q97R Q319R Q358R 1.48 43 Q97R Q319R Q443E 1.21 44 Q97R Q319R Q443R 1.21 45 Q97R Q319R 1.14 50 Q97R Q358R Q443R 1.14 62 Q319E Q443R 1.26 63 Q319E 1.18 64 Q319R Q358E Q443E 1.19 65 Q319R Q358E Q443R 1.28 68 Q319R Q358R Q443R 1.14 70 Q319R Q443E 1.22 73 Q358E Q443E 1.15 74 Q358E Q443R 1.15 75 Q358E 1.18

Example 18 Enzyme Performance

This Example demonstrates that enzyme performance may be affected by charge. Enzyme performance was assessed using heat inactivated detergents as described above in Example 12. Winners were defined as those having Performance Index (PI) a greater than 1. PI is the ratio of mutant residual activity to parent (i.e., S242Q) residual activity. Results are shown in Tables 18-1 and 18-2.

TABLE 18-1 S242Q CCL - CS-28 rice starch microswatch winners, Tide 2x (North American conditions as described in Example 12) Variant # 97 319 358 443 rel charge PI 13 Q97E Q319R Q358R Q443E 0 1.44 14 Q97E Q319R Q358R Q443R 2 1.32 15 Q97E Q319R Q358R 1 1.40 16 Q97E Q319R Q443E −1 1.33 17 Q97E Q319R Q443R 1 1.40 18 Q97E Q319R 0 1.41 20 Q97E Q358E Q443R −1 1.15 23 Q97E Q358R Q443R 1 1.21 25 Q97E Q443E −2 1.18 26 Q97E Q443R 0 1.25 27 Q97E −1 1.16 28 Q97R Q319E Q358E Q443E −2 2.32 29 Q97R Q319E Q358E Q443R 0 2.54 30 Q97R Q319E Q358E −1 2.93 31 Q97R Q319E Q358R Q443E 0 2.27 32 Q97R Q319E Q358R Q443R 2 2.28 33 Q97R Q319E Q358R 1 2.34 34 Q97R Q319E Q443E −1 2.31 35 Q97R Q319E Q443R 1 2.31 36 Q97R Q319E 0 2.14 37 Q97R Q319R Q358E Q443E 0 1.93 38 Q97R Q319R Q358E Q443R 2 1.85 39 Q97R Q319R Q358E 1 2.14 40 Q97R Q319R Q358R Q443E 2 1.92 41 Q97R Q319R Q358R Q443R 4 1.37 42 Q97R Q319R Q358R 3 1.61 43 Q97R Q319R Q443E 1 1.90 44 Q97R Q319R Q443R 3 1.64 45 Q97R Q319R 2 1.99 46 Q97R Q358E Q443E −1 1.40 47 Q97R Q358E Q443R 1 1.29 48 Q97R Q358E 0 1.60 49 Q97R Q358R Q443E 1 1.57 50 Q97R Q358R Q443R 3 1.38 51 Q97R Q358R 2 1.37 52 Q97R Q443E 0 1.51 54 Q97R 1 1.51 55 Q319E Q358E Q443E −3 1.14 56 Q319E Q358E Q443R −1 1.38 57 Q319E Q358E −2 1.10 58 Q319E Q358R Q443E −1 1.25 59 Q319E Q358R Q443R 1 1.41 60 Q319E Q358R 0 1.49 61 Q319E Q443E −2 1.16 62 Q319E Q443R 0 1.45 63 Q319E −1 1.28 64 Q319R Q358E Q443E −1 1.12 65 Q319R Q358E Q443R 1 1.19 66 Q319R Q358E 0 1.36 67 Q319R Q358R Q443E 1 1.24 69 Q319R Q358R 2 1.19 70 Q319R Q443E 0 1.29 76 Q358R Q443E 0 1.22 78 Q358R 1 1.25 79 Q443E −1 1.24 80 Q443R 1 1.17

TABLE 18-2 S242Q CCL - CS-28 rice starch microswatch winners, Persil (Western European conditions) Variant # 97 319 358 443 rel charge PI 2 Q97E Q319E Q358E Q443R −2 1.41 3 Q97E Q319E Q358E −3 1.94 4 Q97E Q319E Q358R Q443E −2 1.61 5 Q97E Q319E Q358R Q443R 0 1.39 6 Q97E Q319E Q358R −1 2.04 7 Q97E Q319E Q443E −3 2.05 8 Q97E Q319E Q443R −1 1.84 9 Q97E Q319E −2 2.27 10 Q97E Q319R Q358E Q443E −2 1.35 13 Q97E Q319R Q358R Q443E 0 1.45 14 Q97E Q319R Q358R Q443R 2 1.17 15 Q97E Q319R Q358R 1 1.22 16 Q97E Q319R Q443E −1 1.26 17 Q97E Q319R Q443R 1 1.29 18 Q97E Q319R 0 1.76 26 Q97E Q443R 0 1.36 27 Q97E −1 1.31 28 Q97R Q319E Q358E Q443E −2 2.21 29 Q97R Q319E Q358E Q443R 0 1.96 30 Q97R Q319E Q358E −1 1.94 31 Q97R Q319E Q358R Q443E 0 2.11 32 Q97R Q319E Q358R Q443R 2 1.87 33 Q97R Q319E Q358R 1 2.41 34 Q97R Q319E Q443E −1 2.20 35 Q97R Q319E Q443R 1 2.21 36 Q97R Q319E 0 2.07 37 Q97R Q319R Q358E Q443E 0 1.86 38 Q97R Q319R Q358E Q443R 2 1.83 39 Q97R Q319R Q358E 1 1.99 40 Q97R Q319R Q358R Q443E 2 1.85 41 Q97R Q319R Q358R Q443R 4 1.36 42 Q97R Q319R Q358R 3 1.90 43 Q97R Q319R Q443E 1 1.99 44 Q97R Q319R Q443R 3 1.94 45 Q97R Q319R 2 1.75 46 Q97R Q358E Q443E −1 1.71 47 Q97R Q358E Q443R 1 1.39 48 Q97R Q358E 0 1.85 50 Q97R Q358R Q443R 3 1.24 51 Q97R Q358R 2 1.36 52 Q97R Q443E 0 1.25 54 Q97R 1 1.88 55 Q319E Q358E Q443E −3 1.12 56 Q319E Q358E Q443R −1 1.17 58 Q319E Q358R Q443E −1 1.16 59 Q319E Q358R Q443R 1 1.25 60 Q319E Q358R 0 1.50 63 Q319E −1 1.36 64 Q319R Q358E Q443E −1 1.10 65 Q319R Q358E Q443R 1 1.18 66 Q319R Q358E 0 1.25 67 Q319R Q358R Q443E 1 1.29 70 Q319R Q443E 0 1.15

Activity was also measured using the BODIPY starch hydrolysis assay as provided herein. The results are shown in Table 18-3. A relative specific activity on this starch substrate (a corn starch) greater than 1 indicates the variant has higher specific activity than the S242Q parent. Relative ppm is the expression titer of the variant relative to the parent, greater than 1 indicates higher titers (in shake tubes) than the S242Q parent.

TABLE 18-3 S242Q CCL - titer and/or BODIPY-starch winners Rel Rel Sp Variant # 97 319 358 443 Charge ppm act 1 Q97E Q319E Q358E Q443E −4 1.27 1.29 2 Q97E Q319E Q358E Q443R −2 1.19 1.31 3 Q97E Q319E Q358E −3 1.00 1.43 4 Q97E Q319E Q358R Q443E −2 1.23 1.43 5 Q97E Q319E Q358R Q443R 0 0.94 1.78 6 Q97E Q319E Q358R −1 0.89 1.81 7 Q97E Q319E Q443E −3 1.40 1.41 8 Q97E Q319E Q443R −1 1.12 1.58 9 Q97E Q319E −2 1.09 1.56 10 Q97E Q319R Q358E Q443E −2 1.45 1.32 11 Q97E Q319R Q358E Q443R 0 1.32 1.49 12 Q97E Q319R Q358E −1 1.58 1.27 13 Q97E Q319R Q358R Q443E 0 0.65 1.44 14 Q97E Q319R Q358R Q443R 2 0.66 1.65 15 Q97E Q319R Q358R 1 0.80 1.64 16 Q97E Q319R Q443E −1 1.09 1.51 17 Q97E Q319R Q443R 1 1.00 1.42 18 Q97E Q319R 0 0.87 1.78 19 Q97E Q358E Q443E −3 1.22 0.88 21 Q97E Q358E −2 1.12 0.88 22 Q97E Q358R Q443E −1 0.91 1.16 23 Q97E Q358R Q443R 1 0.78 1.25 24 Q97E Q358R 0 1.08 1.14 25 Q97E Q443E −2 1.12 1.00 28 Q97R Q319E Q358E Q443E −2 0.78 1.87 29 Q97R Q319E Q358E Q443R 0 0.80 1.81 30 Q97R Q319E Q358E −1 0.68 2.21 31 Q97R Q319E Q358R Q443E 0 0.68 1.96 32 Q97R Q319E Q358R Q443R 2 0.70 2.05 33 Q97R Q319E Q358R 1 0.60 2.27 34 Q97R Q319E Q443E −1 0.65 2.25 35 Q97R Q319E Q443R 1 0.70 2.15 36 Q97R Q319E 0 0.73 2.23 37 Q97R Q319R Q358E Q443E 0 0.93 2.11 38 Q97R Q319R Q358E Q443R 2 0.65 2.21 39 Q97R Q319R Q358E 1 0.82 2.22 40 Q97R Q319R Q358R Q443E 2 0.74 2.28 41 Q97R Q319R Q358R Q443R 4 0.55 2.09 42 Q97R Q319R Q358R 3 0.67 2.48 43 Q97R Q319R Q443E 1 0.84 2.35 44 Q97R Q319R Q443R 3 0.73 2.41 45 Q97R Q319R 2 0.76 2.45 46 Q97R Q358E Q443E −1 0.79 1.45 47 Q97R Q358E Q443R 1 0.75 1.42 48 Q97R Q358E 0 0.82 1.46 49 Q97R Q358R Q443E 1 0.67 1.69 50 Q97R Q358R Q443R 3 0.60 1.60 51 Q97R Q358R 2 0.64 1.29 52 Q97R Q443E 0 0.83 1.43 54 Q97R 1 0.72 1.49 55 Q319E Q358E Q443E −3 0.99 1.15 56 Q319E Q358E Q443R −1 0.77 1.40 57 Q319E Q358E −2 0.83 1.34 58 Q319E Q358R Q443E −1 0.73 1.49 59 Q319E Q358R Q443R 1 0.67 1.61 60 Q319E Q358R 0 0.80 1.67 61 Q319E Q443E −2 0.91 1.39 62 Q319E Q443R 0 0.73 1.45 63 Q319E −1 0.75 1.41 64 Q319R Q358E Q443E −1 1.05 1.28 65 Q319R Q358E Q443R 1 0.94 1.42 66 Q319R Q358E 0 0.96 1.39 67 Q319R Q358R Q443E 1 1.02 1.50 68 Q319R Q358R Q443R 3 0.71 1.57 69 Q319R Q358R 2 0.71 1.58 70 Q319R Q443E 0 0.91 1.49 72 Q319R 1 0.95 1.56 77 Q358R Q443R 2 0.67 1.22 78 Q358R 1 0.66 1.15

Example 19 Balancing Mutational Effects on Amylase Activity and Expression

This Example illustrates that two conflicting enzyme properties can be simultaneously optimized by the introduction of multiple amino acid substitutions.

As determined during development of the present invention, the median expression of AmyS-242Q decreased with increasing positive charge. However, specific BODIPY starch hydrolysis increased with increasing positive charge. Enhanced recombinant amylase expression and starch hydrolysis are desirable in an engineered variant of AmyS-242Q suitable for starch liquefaction in the fuel ethanol industry or cleaning in detergent applications for instance. These properties, however, are apparently conflicting properties. As determined during development of the present invention, using the methods of the present invention, it is possible to produce a more highly expressed amylase variant without severely compromising starch hydrolysis by selectively combining single mutations. The strategy described herein was successfully used to produce and select multiply-substituted AmyS-242Q variants having improvements in a first property (e.g., expression as the primary property), while improving or not sacrificing a second property (e.g., starch hydrolysis as the secondary property).

In addition, in converse to median expression of AmyS-242Q variants, corn starch microswatch cleaning increased with increasing positive charge. Enhanced recombinant amylase expression and cleaning performance are desirable in an engineered variant of AmyS-242Q. These properties, however, are also apparently conflicting properties. As determined during development of the present invention, using the methods of the present invention, it is possible to produce a more highly expressed amylase variant without severely compromising cleaning performance by selectively combining single mutations. The strategy described herein was successfully used to produce and select multiply-substituted AmyS-242Q variants having improvements in a first property (e.g., expression as the primary property), while improving or not sacrificing a second property (e.g., rice starch microswatch cleaning as the secondary property).

In particular, an eighty member AmyS-S242Q charge combinatorial library (CCL) comprising variants having combinations of from one to four substitutions of charged residues was tested for shake tube expression, BODIPY-starch hydrolysis, and rice starch cleaning activity. AmyS-S242Q winners are shown in Tables 19-1 and 19-2. Importantly, the multiply-substituted variants of Table 19-1 have equal or improved expression and equal or improved BODIPY-starch hydrolysis as compared to the parent enzyme. Similarly, the multiply-substituted variants of Table 19-2 have equal or improved expression and equal or improved rice starch cleaning activity as compared to the parent enzyme.

TABLE 19-1 AmyS-S242Q Expression and BODIPY-Starch Hydrolysis Winners Expres- Vari- sion BODIPY ant 97 319 358 443 Charge (PI) (PI) 1 Q97E Q319E Q358E Q443E −4 1.27 1.29 2 Q97E Q319E Q358E Q443R −2 1.19 1.31 3 Q97E Q319E Q358E −3 1.00 1.43 4 Q97E Q319E Q358R Q443E −2 1.23 1.43 7 Q97E Q319E Q443E −3 1.40 1.41 8 Q97E Q319E Q443R −1 1.12 1.58 9 Q97E Q319E −2 1.09 1.56 10 Q97E Q319R Q358E Q443E −2 1.45 1.32 11 Q97E Q319R Q358E Q443R 0 1.32 1.49 12 Q97E Q319R Q358E −1 1.58 1.27 16 Q97E Q319R Q443E −1 1.09 1.51 17 Q97E Q319R Q443R +1 1.00 1.42 24 Q97E Q358R 0 1.08 1.14 25 Q97E Q443E −2 1.12 1.00 64 Q319R Q358E Q443E −1 1.05 1.28 67 Q319R Q358R Q443E +1 1.02 1.50

TABLE 19-2 AmyS-S242Q Expression and Rice-Starch Hydrolysis Winners Expres- Variant 97 319 358 443 Charge sion CS-28 1 Q97E Q319E Q358E Q443E −4 1.27 1.01 11 Q97E Q319R Q358E Q443R 0 1.32 1.18 12 Q97E Q319R Q358E −1 1.58 1.13 16 Q97E Q319R Q443E −1 1.09 1.43 17 Q97E Q319R Q443R +1 1.00 1.55 24 Q97E Q358R 0 1.08 1.15 25 Q97E Q443E −2 1.12 1.09 64 Q319R Q358E Q443E −1 1.05 1.18 67 Q319R Q358R Q443E +1 1.02 1.15

In sum, because enzyme activity and enzyme production have different charge dependencies (see FIGS. 27A, 27B, 28A, and 28B) they are negatively correlated (See FIGS. 26A and 26B). However, there are a number of variants that are improved in both expression and activity, and analyzing the library in this manner allows them to be identified.

Although demonstrated with amylases this method is applicable to other enzyme classes such as proteases, lipases, cellulases, transferases and pectinases. Moreover any combination of two or more properties can be analyzed simultaneously such as expression, activity, binding, thermal stability, detergent and chelant stability.

Example 20 Microswatch Cleaning and Starch Hydrolysis

Enzyme performance was assessed using heat inactivated detergents as described above. Assays were performed as described above in Example 12 (see Rice Starch Microswatch Assay for testing Amylase Performance and Bodipy-Starch Assay For Determination Of Amylase Activity). Winners are defined as those having Performance Index (PI) a greater than 1. PI is the ratio of mutant residual activity to WT residual activity. Table 20-1 shows the calculations for AmyS variants that are better than wild type (ΔΔG<0) compared to the charge change scores (Δ CHRG). The charge change, Kyte-Doolittle, Eisenberg and hydrogen bonding are defined in WO 2008/153925, filed 6 Jun. 2008. In addition Table 20-1 shows the results for Kyte-Doolittle hydropathicity (Δ K-D) and Eisenberg hydrophobicity scales (Δ E). Table 20-1 also shows values for hydrogen bonding (Δ HB), with a score of −2 meaning the loss of hydrogen bonding ability. Table 20-1 shows the calculations for AmyS variants that are better than wild type for corn flour hydrolysis at 5, 10, and 60 min (CF5, CF10, CF60), activity on DP7 substrates at pH 4.0 and 5.8 (pH 4, pH 5.8), rice starch cleaning at pH 8.6 and 10 (Clean 8 and Clean 10), and protein expression in B. subtilis (EXP). The effect of charge on activity has the opposite direction to the effect of charge on expression. Hydrogen bonding and hydrophobicity also demonstrate statistically relevant effects on these properties. Clearly, properties of amino acid substitutions such as charge and hydrophobicity can affect expression levels in B. subtilis and E. coli, as well as basic activity and stability of proteins.

TABLE 20-1 AmyS Quintiles for Multiple Properties AmyS o/e AmyS o/e AmyS o/e AmyS o/e CF5 ΔΔG CF5 ΔΔG CF5 ΔΔG CF5 ΔΔG Δ CHRG −2 1.60 Δ HB −2 0.84 Δ K-D −2 1.12 Δ E −2 1.13 Δ CHRG −1 1.29 Δ HB −1 0.98 Δ K-D −1 1.19 Δ E −1 1.09 Δ CHRG 0 0.97 Δ HB 0 1.02 Δ K-D 0 0.83 Δ E 0 1.05 Δ CHRG +1 0.84 Δ HB +1 0.92 Δ K-D +1 1.15 Δ E +1 0.89 Δ CHRG +2 0.56 Δ HB +2 1.19 Δ K-D +2 0.77 Δ E +2 1.12 CF10 ΔΔG CF10 ΔΔG CF10 ΔΔG CF10 ΔΔG Δ CHRG −2 1.66 Δ HB −2 0.86 Δ K-D −2 1.10 Δ E −2 1.26 Δ CHRG −1 1.18 Δ HB −1 1.00 Δ K-D −1 1.15 Δ E −1 1.04 Δ CHRG 0 0.97 Δ HB 0 1.02 Δ K-D 0 0.86 Δ E 0 1.08 Δ CHRG +1 0.91 Δ HB +1 0.97 Δ K-D +1 1.12 Δ E +1 0.90 Δ CHRG +2 0.77 Δ HB +2 1.12 Δ K-D +2 0.82 Δ E +2 1.16 CF60 ΔΔG CF60 ΔΔG CF60 ΔΔG CF60 ΔΔG Δ CHRG −2 1.46 Δ HB −2 1.00 Δ K-D −2 0.94 Δ E −2 0.98 Δ CHRG −1 1.33 Δ HB −1 0.96 Δ K-D −1 1.15 Δ E −1 1.01 Δ CHRG 0 0.96 Δ HB 0 1.01 Δ K-D 0 0.79 Δ E 0 1.05 Δ CHRG +1 0.84 Δ HB +1 0.95 Δ K-D +1 1.16 Δ E +1 0.94 Δ CHRG +2 0.82 Δ HB +2 1.05 Δ K-D +2 0.89 Δ E +2 1.54 pH 4 ΔΔG pH 4 ΔΔG pH 4 ΔΔG pH 4 ΔΔG Δ CHRG −2 1.63 Δ HB −2 0.91 Δ K-D −2 1.29 Δ E −2 1.07 Δ CHRG −1 1.28 Δ HB −1 0.89 Δ K-D −1 1.19 Δ E −1 1.13 Δ CHRG 0 0.96 Δ HB 0 0.97 Δ K-D 0 0.72 Δ E 0 1.01 Δ CHRG +1 0.88 Δ HB +1 0.93 Δ K-D +1 1.12 Δ E +1 0.89 Δ CHRG +2 0.19 Δ HB +2 1.26 Δ K-D +2 0.86 Δ E +2 0.95 pH 5.8 ΔΔG pH 5.8 ΔΔG pH 5.8 ΔΔG pH 5.8 ΔΔG Δ CHRG −2 1.66 Δ HB −2 0.99 Δ K-D −2 1.00 Δ E −2 1.23 Δ CHRG −1 1.26 Δ HB −1 0.99 Δ K-D −1 1.17 Δ E −1 1.06 Δ CHRG 0 0.95 Δ HB 0 0.95 Δ K-D 0 0.80 Δ E 0 0.99 Δ CHRG +1 0.94 Δ HB +1 0.90 Δ K-D +1 1.08 Δ E +1 0.94 Δ CHRG +2 0.83 Δ HB +2 1.15 Δ K-D +2 0.92 Δ E +2 1.16 Clean8 ΔΔG Clean8 ΔΔG Clean8 ΔΔG Clean8 ΔΔG Δ CHRG −2 1.34 Δ HB −2 1.07 Δ K-D −2 0.89 Δ E −2 0.88 Δ CHRG −1 1.22 Δ HB −1 1.02 Δ K-D −1 1.10 Δ E −1 0.98 Δ CHRG 0 0.96 Δ HB 0 0.96 Δ K-D 0 0.83 Δ E 0 1.00 Δ CHRG +1 0.94 Δ HB +1 0.90 Δ K-D +1 1.07 Δ E +1 1.01 Δ CHRG +2 0.62 Δ HB +2 1.05 Δ K-D +2 1.02 Δ E +2 1.32 Clean10 ΔΔG Clean10 ΔΔG Clean10 ΔΔG Clean10 ΔΔG Δ CHRG −2 1.32 Δ HB −2 0.86 Δ K-D −2 1.03 Δ E −2 0.81 Δ CHRG −1 1.43 Δ HB −1 1.36 Δ K-D −1 1.11 Δ E −1 1.03 Δ CHRG 0 0.92 Δ HB 0 0.72 Δ K-D 0 0.80 Δ E 0 1.00 Δ CHRG +1 0.88 Δ HB +1 1.07 Δ K-D +1 1.16 Δ E +1 0.97 Δ CHRG +2 0.74 Δ HB +2 1.11 Δ K-D +2 0.91 Δ E +2 1.48 EXP ΔΔG EXP ΔΔG EXP ΔΔG EXP ΔΔG Δ CHRG −2 0.00 Δ HB −2 0.63 Δ K-D −2 0.65 Δ E −2 0.71 Δ CHRG −1 0.35 Δ HB −1 0.91 Δ K-D −1 1.11 Δ E −1 1.29 Δ CHRG 0 1.08 Δ HB 0 0.95 Δ K-D 0 1.49 Δ E 0 1.06 Δ CHRG +1 1.35 Δ HB +1 1.39 Δ K-D +1 0.77 Δ E +1 0.79 Δ CHRG +2 1.64 Δ HB +2 1.16 Δ K-D +2 0.72 Δ E +2 0.20

Example 21 Modulating of an Enzyme's pH-Activity Profile

This Example describes the use of surface charge mutations to optimize an enzyme's pH-activity profile for a given reaction. FIG. 31A shows rice starch microswatch cleaning activity as a function of pH for the first AmyS charge ladder of Example 15. The pH range from 3.0 to 4.25 was in 200 mM Na formate containing 0.01% Tween-80, while the pH range from 4.25 to 5.5 was in 200 mM Na acetate containing 0.01% Tween-80. The data are fit to titration curves, each with a single pKa value. FIG. 31B show an apparent pKa for AmyS catalysis as a function of charge change for the first AmyS charge ladder of Example 15. These data demonstrate that pH-activity profiles for an α-amylase can be significantly shifted by surface charge mutations, even in 200 mM buffer. Although this had been reported at very low ionic strength for subtilisin (Russell et al. (1987) J Mol. Biol. 193: 803-13) and for D-xylose isomerase (Cha et al. (1998) Mol. Cell. 8:374-82), this is believed to be the first time this has been accomplished with α-amylase, and, surprisingly, even at high ionic strength.

Example 22 AmyS Superscreen

The following assays were used in the examples described below. Any deviations from the protocols provided below are indicated in the examples. In these experiments, a 96 well spectrophotometer was used to measure the absorbance of the products formed after the completion of the reactions.

Starch Hydrolysis Assay for Specific Activity Determination and Thermal Stability

α-amylase activity assay on corn flour was performed to measure specific activity and stability of B. subtilis AmyS and AmyS variants. Conditions that closely mimic real-world applications in cleaning and grain processing were used. Activity is defined as reducing ends generated due to the enzymatic breakdown of corn flour, determined by the PAHBAH (p-hydroxybenzoic acid hydrazide) method. Stability is defined as sustained activity at 85° C.

Hardware: Inheco Variomag Teleshake 95 with PCR plate adapter, Thermo Electron Multidrop, Axygen PCR-96-FS-C full-skirt PCR plate, Thermocyclers—with a minimum of 4 96-well blocks (an MJ Research Tetrad), Biomek FX liquid handlers.

Starch Hydrolysis: Azure Farms Organic Corn Flour, sifted for liquid-handling purposes was used to obtain the <600 micron fraction, baked 4 hours at 80° C., then allowed to equilibrate overnight at room temperature. A 2% w/w suspension was prepared in 500-g and 1000-g batches. The suspension was stirred vigorously and continuously during pH adjustment, pH equilibration, and transfer from beaker to PCR plate. For 1,000 g, 23 g pre-baked corn flour and 977 g house deionized water were stirred for 15 minutes, adjusted with H2SO4 to pH 5.8, and allowed to equilibrate for 30 minutes, at which point a final pH adjustment was performed if necessary. 8-channel pipets bearing tips trimmed to an opening size of approximately 1.5 mm were used to deliver the suspension into the wells of Axygen PCR plates.

Culture supernatants of AmyS and AmyS variants were diluted to approximately 1 μg/mL in dilution buffer (water+0.005% Tween-80) and 10 μL diluted supernatant each were transferred to the 5-minute, 10-minute and 60-minute reaction plates and mixed once by pipetting sample up and down. An aliquot of 50 μL light mineral oil was transferred to each well. Plates were transferred to the Inheco units pre-heated to 85° C. At the indicated time points following incubation (5, 10 and 60 minutes), the starch hydrolysis reaction was stopped by addition of 10 μL of 4N NaOH to each well. The starch hydrolysis reaction products were analyzed by the PAHBAH assay.

PAHBAH assay: Aliquots of 80 μL of 0.5 N NaOH were added to all wells of an empty PCR plate followed by 20 μL of PAHBAH reagent (5% w/v p-hydroxybenzoic acid hydrazide (PAHBAH, Sigma # H9882, dissolved in 0.5 N HCl) and mixed by pipetting up and down (PAHBAH reaction plate). 10 μL of the starch hydrolysis reaction supernatants were added to the PAHBAH reaction plate. All plates were sealed and placed in the thermocycler, programmed for 2 minutes at 95° C., and then cooled to 20° C. Samples of 80 μL of the developed PAHBAH reaction mixtures were transferred to a fresh (read) plate and absorbance was measured at 405 nm in a spectrophotometer.

Cleaning Swatch Assay for Stain Removal Performance

In this assay, the stain removal performance of B. subtilis AmyS and AmyS variants was determined in a microtiter plate scale using CS-28 rice starch stain microswatches. Microswatches of ¼″ circular diameter were obtained from CFT Vlaardingen (Netherlands). Two microswatches were placed into each well of a 96-well microtiter plate.

The filtered culture broth samples were tested at an appropriate concentration by dilution with a mixture of 10 mM NaCl, 0.1 mM CaCl₂, 0.005% Tween-80 to 20× the desired final concentration in the performance test (final conc. in test 0.025-0.10 ppm).

Amylase performance was measured at both pH 8 and pH 10.

Either 190 μl of buffer solution, containing 25 mM HEPES (Sigma, H7523), 2 mM CaCl₂, 0.005% Tween-80, pH 8.0, or 190 μl of buffer solution, containing 25 mM CAPS (Sigma, C2632), 2 mM CaCl₂, 0.005% Tween-80, pH 10.0 were added to each well of the plates containing microswatches. 10 μL of diluted amylase samples were added to each microswatch containing well (to provide a total volume of 200 μL/well). The plate was covered with a plate seal and placed in an incubator for 60 minutes at 40° C., with agitation at 1150 rpm (iEMS incubator). Following incubation under the appropriate conditions, 100 μL of solution from each well was removed, placed into a fresh microtiter plate and absorbance was measured at 488 nm in a spectrophotometer. “Blank controls”, containing 2 microswatches per well and detergent but no amylase samples were also included in the test.

Calculation of the CS-28 rice starch hydrolysis performance: The obtained absorbance value was corrected for the blank value (microswatches incubated in the absence of enzyme). The resulting absorbance—ΔOD₄₈₈—was a measure for the amylolytic activity. For each sample (AmyS or AmyS variant) the performance index was calculated by dividing the activity of the variant by the activity of the wildtype enzyme. The performance index compared the performance of the variant (actual value) and the standard AmyS reference enzyme (theoretical value) at the same protein concentration.

A performance index (PI) that is greater than 1 (PI>1) identified a better variant (as compared to the standard, e.g., wild-type), while a PI of 1 (PI=1) identified a variant that performs the same as the standard, and a PI that is less than 1 (PI<1) identified a variant that performs worse than the standard. Thus, the PI identified variants with performance differences over the wildtype enzyme.

The following site variants were evaluated using the assays described in this Example:

P17A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y

D19A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y

T21A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y

N28A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y

S51A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y

G72A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y

V74A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y

A82A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y

Q86A, C, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y

Q89A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, Y

A93A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y

W115D, E, F, G, K, L, N, P, Q, R, S, V, Y

D117A, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W

P123A, D, E, G, K, L, M, P, Q, R, S, T, V

S124A, C, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, Y

D125A, D, E, G, K, M, Q, R, S, T, V

N127A, C, D, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, Y

I130A, G, H, I, K, L, M, N, P, Q, R, S, T, V, W

G132A, C, D, E, F, G, H, L, M, N, P, R, S, T, V, W, Y

Q135A, F, G, K, L, M, P, Q, R, S, T, V, Y

P145A, D, E, F, H, I, K, L, N, P, R, S, T, V, Y

G146A, C, D, E, G, H, K, L, P, R, S, T, V, W

G148A, C, D, E, F, G, H, L, N, P, Q, R, S, T, V, W, Y

S153A, C, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y

Y159A, C, D, E, F, G, H, K, L, N, R, S, T, V, W

W166C, E, F, G, H, I, K, L, M, P, R, S, T, V, Y

S169A, C, D, E, F, G, I, K, L, M, N, P, Q, R, T, V, Y

K171C, D, E, G, H, K, L, M, P, Q, R, S, T, V, W, Y

R179A, G, H, L, M, P, Q, R, S, T, V, W, Y

G180A, C, D, F, G, H, I, K, L, N, P, R, S, T, V, Y

I181A, C, D, E, F, G, H, I, K, L, P, R, S, T, V, Y

G182A, C, D, E, F, G, H, K, L, P, R, S, T, V, Y

K183A, C, E, F, G, H, K, L, M, P, Q, R, S, T, V, W, Y

W187A, C, E, G, I, K, L, N, P, Q, R, S, V, W

G194A, E, G, H, K, L, M, P, R, S, T, V, W

P209A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y

N224A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y

S242A, C, D, G, I, K, L, M, Q, R, S, T, V

P245A, C, D, E, F, H, I, L, M, N, P, Q, R, S, T, V, Y

G256A, C, D, E, G, H, I, K, L, M, N, P, R, S, T, V, W

D269A, C, D, F, G, H, I, K, M, N, P, Q, R, S, T, Y

N271A, D, F, H, I, K, L, M, N, P, S, T, V, W, Y

T278A, E, G, H, I, K, L, M, N, P, R, S, T, W, Y

N281A, D, G, H, I, L, M, N, P, Q, R, S, T, V, Y

G302C, D, E, F, G, H, I, L, M, N, P, R, S, T, V, W, Y

A304A, D, E, F, H, L, M, N, P, R, S, T, V, W, Y

R308A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, Y

T321A, C, F, H, I, L, P, Q, R, S, T, V, Y

Q358A, C, D, E, F, G, H, L, M, N, P, Q, R, S, T, V

P378C, D, F, G, H, I, L, N, P, R, S, T, V, Y

S382A, C, D, E, G, H, I, K, L, M, N, P, R, S, T, V, W

K383A, C, D, E, F, H, K, L, M, N, P, Q, R, S, T, W, Y

T398A, C, D, E, I, K, L, M, N, P, Q, R, S, T, V

H405A, C, D, F, G, H, K, L, M, N, P, Q, R, S, T, W, Y

T417A, D, E, H, I, L, M, P, Q, R, S, T, V, W

E418A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, Y

P420A, C, D, E, H, I, L, M, N, P, R, S, T, V, W, Y

G421A, D, E, F, G, H, I, L, N, P, Q, R, S, T, W, Y

P432A, D, E, H, K, L, M, N, P, Q, R, S, T, Y

W437C, D, E, F, G, H, L, M, N, Q, R, S, T, V, W, Y

Q443A, C, F, G, K, L, N, P, Q, R, S, T, V, W, Y

G446A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y

G454A, C, D, E, G, H, I, K, L, M, N, P, R, S, T, V

S457A, C, D, E, G, H, K, L, M, N, P, Q, R, S, T, V, W, Y

T459A, D, G, I, K, L, Q, R, S, T, V, Y

T461A, D, E, F, G, I, K, L, N, P, R, S, T, V, W, Y

S464D, E, G, H, I, K, L, M, N, P, Q, S, V, W, Y

G474A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V

R483A, C, F, G, K, L, M, N, P, Q, R, S, T, V, Y

Example 23 Performance of AmyS Variants

The performance of AmyS variants (e.g., as described in Example 22) were tested for protein expression (expression), hydrolysis of corn flour for 10 minutes (Corn Flour 10) or hydrolysis of corn flour for 60 minutes (Corn Flour 60), activity on DP7 substrate at pH 4 (DP7 pH 4) or activity on DP7 substrate at pH5.8 (DP7 pH 5.8), and CS 28 rice starch stained microswatch cleaning at pH 8 (cleaning pH 8) or CS 28 rice starch stained microswatch cleaning at pH 10 (cleaning 10). The results are shown in Table 23-1. Protein expression was measured by Bradford assay described in Example 12. The corn flour hydrolysis and cleaning swatch assays were performed as described in Example 22. Functionality of AmyS variants was quantified as a performance index (Pi) (i.e., the ratio of performance of a variant relative to wild type AmyS). A PI>1 for any property indicates that the variant is improved (compared to the control) for that property. ND indicates that the value obtained was outside the range of the assay.

TABLE 23-1 Performance of AmyS variants Corn Corn Flour Flour Cleaning Cleaning Position variant 10 60 DP7 pH 4 DP7 pH 5.8 pH 8 pH 10 Expression 017 P017A 1.08 1.07 1.13 1.32 1.35 1.04 0.58 017 P017C 1.38 1.46 1.20 1.41 1.47 1.29 0.50 017 P017D 1.30 1.30 1.02 1.24 1.33 1.10 0.58 017 P017E 1.07 1.20 1.03 1.18 1.29 1.04 0.70 017 P017F 0.95 1.10 0.84 0.95 1.37 0.83 0.59 017 P017G 0.90 0.83 0.83 0.90 1.04 0.84 0.88 017 P017H 0.91 0.84 1.03 1.03 1.07 0.86 0.96 017 P017I 0.78 0.83 0.79 0.91 0.83 0.77 0.87 017 P017K 0.88 0.64 0.83 0.96 1.11 0.88 0.90 017 P017L 0.69 0.64 0.44 0.32 0.91 0.92 0.67 017 P017M 1.19 1.46 1.13 1.32 1.58 0.83 0.50 017 P017N 1.05 1.16 1.13 1.30 1.14 0.81 0.70 017 P017Q 1.24 1.31 1.19 1.21 1.09 0.90 0.73 017 P017R 1.21 1.23 0.93 1.13 1.40 1.01 0.71 017 P017S 0.97 0.85 0.78 0.84 1.00 0.87 0.76 017 P017T 0.81 0.91 0.68 0.75 1.12 0.73 0.76 017 P017V 0.79 0.79 0.70 0.75 0.94 0.82 0.81 017 P017W 0.81 0.77 0.70 0.82 0.89 0.77 0.75 017 P017Y 0.75 0.79 0.98 0.97 1.01 0.95 0.96 019 D019A 1.50 1.69 1.29 1.55 2.01 1.42 0.48 019 D019C 1.34 1.49 1.29 1.47 1.59 1.25 0.51 019 D019E 1.39 1.38 1.41 1.48 1.69 1.40 0.67 019 D019F 3.22 3.51 0.75 0.87 6.54 3.43 0.10 019 D019G 1.20 1.29 1.19 1.20 0.98 1.22 0.75 019 D019H 0.93 0.97 0.91 1.05 1.32 0.98 0.93 019 D019I 1.13 0.90 0.93 1.05 1.82 0.96 0.38 019 D019K 0.90 0.81 0.97 1.09 0.93 0.91 0.99 019 D019L 0.84 0.65 −0.65 −0.23 1.79 1.94 0.13 019 D019M 1.60 1.98 1.07 1.40 1.63 1.41 0.35 019 D019N 1.34 1.16 1.69 1.23 1.20 1.06 0.71 019 D019P 0.93 1.14 1.00 1.27 1.56 1.32 0.53 019 D019Q 1.35 1.24 1.94 1.65 1.31 1.20 0.74 019 D019R 1.02 1.05 0.94 1.03 1.44 1.12 0.80 019 D019S 1.03 1.12 1.08 1.12 1.09 0.89 0.95 019 D019T 1.04 1.06 1.07 1.11 1.22 0.89 0.97 019 D019V 1.05 1.28 1.07 1.27 2.16 1.52 0.36 019 D019W 0.64 0.93 0.52 0.71 1.24 0.81 0.56 019 D019Y 0.98 1.10 1.43 1.32 1.34 1.03 0.69 021 T021A 1.25 1.34 1.36 1.50 1.41 1.19 0.75 021 T021C 1.59 1.73 1.42 1.58 1.79 1.26 0.49 021 T021D 1.23 1.39 1.63 1.54 1.48 1.27 0.78 021 T021E 1.32 1.35 1.53 1.59 1.48 1.05 0.72 021 T021F 1.26 1.36 1.66 1.48 1.42 1.11 0.71 021 T021G 1.11 1.14 1.35 1.29 1.24 1.05 0.91 021 T021H 0.87 0.85 0.95 0.98 1.08 0.86 1.10 021 T021I 1.04 1.04 1.31 1.31 1.57 1.14 0.71 021 T021K 0.89 0.88 1.02 1.07 1.02 1.01 1.07 021 T021L 0.80 0.92 1.33 1.20 1.08 0.88 0.89 021 T021M 1.37 1.40 1.34 1.55 1.48 1.34 0.75 021 T021N 1.36 1.42 1.28 1.47 1.23 1.16 0.75 021 T021P 1.13 1.25 1.14 1.27 1.30 1.20 0.82 021 T021Q 1.32 1.42 1.50 1.55 1.33 1.20 0.79 021 T021R 1.17 1.26 1.14 1.21 1.23 1.11 0.86 021 T021S 1.08 1.28 1.09 1.17 1.12 0.97 0.91 021 T021V 1.10 1.19 1.12 1.24 1.35 0.95 0.73 021 T021W 0.98 1.01 0.91 0.95 1.17 1.11 0.75 021 T021Y 0.81 0.89 1.02 1.07 1.03 0.62 0.87 028 N028A 1.28 1.51 1.24 1.42 1.64 1.39 0.74 028 N028C −0.92 −2.93 1.18 0.31 1.18 2.17 −0.05 028 N028D 1.29 1.39 1.69 1.63 1.37 1.23 0.77 028 N028E 1.26 1.36 1.21 1.38 1.21 1.06 0.79 028 N028F 1.29 1.34 1.16 1.35 1.48 1.26 0.54 028 N028G 0.98 1.05 0.99 1.08 0.98 0.81 1.06 028 N028H 0.98 1.09 1.06 1.18 1.20 1.01 0.94 028 N028I 0.88 1.02 0.86 0.97 1.17 0.87 0.71 028 N028K 0.93 0.95 1.01 1.09 0.84 0.94 0.98 028 N028L 0.79 1.00 0.91 1.02 1.11 0.87 0.74 028 N028M 1.48 1.67 1.62 1.79 2.01 1.57 0.53 028 N028P 1.47 1.60 2.00 2.47 1.89 1.47 0.48 028 N028Q 1.19 1.23 1.01 1.18 1.20 1.24 0.75 028 N028R 1.11 1.07 1.10 1.27 1.38 1.10 0.80 028 N028S 1.11 1.23 0.98 1.08 1.03 0.94 0.91 028 N028T 1.13 1.22 1.07 1.21 1.22 0.99 0.84 028 N028V 0.98 0.97 1.12 1.13 1.35 0.90 0.75 028 N028W 1.00 1.05 0.92 1.08 1.25 0.92 0.65 028 N028Y 0.92 1.00 0.87 0.88 1.13 0.76 0.86 051 S051A 1.01 0.99 1.39 1.21 1.11 1.14 0.97 051 S051C 0.96 1.01 1.06 1.36 0.91 0.51 0.80 051 S051D 0.94 1.00 1.34 1.42 0.95 1.07 0.97 051 S051E 0.71 0.64 1.10 1.25 0.79 1.02 1.11 051 S051F 0.96 0.93 1.04 1.29 1.06 1.06 0.89 051 S051G 0.83 0.77 0.81 0.82 0.80 1.09 1.24 051 S051H 0.74 0.70 0.56 0.86 0.93 0.99 1.20 051 S051I 0.75 0.73 0.80 0.83 0.82 0.74 1.18 051 S051K 0.62 0.58 0.54 0.67 0.68 0.82 1.39 051 S051L 0.71 0.72 0.85 0.88 0.74 0.83 1.41 051 S051M 0.97 1.00 1.24 1.26 1.11 1.01 0.94 051 S051N 1.04 1.05 0.99 1.02 0.64 1.02 0.96 051 S051P 0.81 0.79 0.31 1.23 1.22 0.78 0.75 051 S051Q 1.01 0.96 1.07 1.08 1.07 1.06 1.03 051 S051R 0.88 0.88 0.66 0.89 0.92 1.09 1.02 051 S051T 0.89 0.80 0.76 0.79 0.96 0.91 1.17 051 S051V 0.78 0.70 0.70 0.75 0.86 0.78 1.21 051 S051W 0.81 0.76 0.81 0.94 0.87 0.81 1.17 051 S051Y 0.70 0.76 0.98 0.96 0.83 0.93 1.16 072 G072A 1.53 1.40 1.25 1.36 1.46 1.46 0.65 072 G072C 1.53 1.39 1.52 1.68 1.40 1.20 0.61 072 G072D 1.36 1.38 1.69 1.72 1.36 1.59 0.78 072 G072E 0.13 0.59 0.75 0.39 −0.18 −0.75 −0.19 072 G072F 0.07 0.74 1.79 0.84 0.39 0.30 −0.07 072 G072H 0.97 0.96 0.95 1.09 1.05 1.11 0.98 072 G072I 12.27 −18.04 −7.88 −5.43 −47.34 −40.91 0.00 072 G072K 0.20 −2.21 −4.25 −2.09 3.20 −0.46 0.04 072 G072L 0.21 −1.59 −3.13 −1.59 −0.59 −0.13 0.04 072 G072M 0.09 0.37 0.78 0.27 0.06 0.01 −0.16 072 G072N −0.09 0.41 0.34 0.12 0.01 0.12 −0.17 072 G072P −0.20 0.29 1.24 0.48 0.06 −0.23 −0.11 072 G072Q 1.68 1.60 1.60 1.66 1.62 1.60 0.68 072 G072R 1.23 1.19 0.83 1.05 1.06 1.42 0.80 072 G072S 0.77 −1.60 −0.59 2.54 0.70 8.99 −0.01 072 G072T 0.93 0.98 0.88 0.94 1.08 1.05 1.02 072 G072V 1.31 1.27 1.19 1.30 1.48 1.22 0.68 072 G072W 0.10 −0.61 −1.03 −0.54 0.41 −0.49 0.12 072 G072Y 1.11 1.01 1.28 1.31 1.21 1.01 0.84 074 V074A 1.43 1.43 1.55 1.54 1.43 1.38 0.79 074 V074C 0.05 0.20 0.77 0.33 −0.16 0.40 −0.19 074 V074D −0.22 0.91 2.69 1.24 0.32 −0.58 −0.05 074 V074E 1.65 1.71 1.69 1.69 1.56 1.84 0.65 074 V074F 2.44 2.48 0.48 0.33 3.48 3.18 0.13 074 V074G 1.29 1.28 1.15 1.14 0.98 1.08 0.97 074 V074H 0.79 0.78 0.82 0.89 0.84 0.78 1.19 074 V074I 1.15 1.16 1.26 1.26 1.21 1.01 0.98 074 V074K 0.08 −0.61 −1.67 −0.73 1.78 0.54 0.07 074 V074L 0.77 −0.57 −2.18 −1.10 0.59 −1.43 0.07 074 V074M −0.14 −0.19 1.02 0.48 0.45 −0.53 −0.13 074 V074N −0.22 0.25 1.07 0.53 0.05 0.15 −0.13 074 V074Q 1.57 1.60 1.61 1.59 1.30 1.16 0.78 074 V074R −0.93 −0.49 1.45 0.77 −0.63 −1.66 −0.08 074 V074S −3.20 −3.28 −0.69 −1.93 −2.28 −5.92 0.05 074 V074T 7.70 −8.69 −7.16 −3.32 −0.73 −6.93 0.02 074 V074W 0.47 −0.38 −3.18 −1.38 0.49 0.72 0.04 074 V074Y 1.12 1.08 0.88 0.91 0.97 1.00 0.93 082 A082C 1.45 1.58 1.16 1.26 1.31 1.25 0.64 082 A082E 1.37 1.32 1.36 1.39 1.12 1.02 0.89 082 A082F 1.36 1.33 1.07 1.14 1.25 1.19 0.84 082 A082G 1.17 1.33 0.79 0.93 1.18 1.07 0.68 082 A082H 1.08 1.04 0.95 0.96 0.90 1.13 1.15 082 A082I 0.96 1.00 1.04 1.03 0.97 0.82 1.15 082 A082K 9.74 1.65 −13.38 −6.33 −9.02 35.95 0.01 082 A082L 1.00 0.96 0.94 0.99 0.82 0.84 1.08 082 A082M −0.42 0.35 0.64 0.22 0.16 −0.23 −0.20 082 A082N 1.36 1.39 1.35 1.45 1.38 1.31 0.83 082 A082P 1.54 1.45 1.25 1.38 1.44 1.14 0.76 082 A082Q −0.19 0.28 1.16 0.54 0.15 1.37 −0.12 082 A082R 1.17 1.17 1.29 1.34 1.42 1.45 0.99 082 A082S 1.02 1.07 0.92 1.01 0.90 0.86 1.06 082 A082T 1.08 1.08 0.99 1.06 1.00 1.32 1.02 082 A082V 0.98 1.08 0.97 1.05 1.02 0.90 1.02 082 A082W 1.16 1.16 0.83 0.99 1.08 0.97 0.96 082 A082Y 0.81 0.87 0.95 0.95 0.95 1.06 1.14 086 Q086A 1.00 1.11 1.43 1.46 1.25 1.02 0.83 086 Q086C 1.00 1.11 1.01 1.19 1.09 0.64 0.73 086 Q086D 0.96 1.03 1.22 1.29 1.11 0.99 0.87 086 Q086E 0.92 0.93 1.12 1.15 0.80 0.97 0.97 086 Q086F 0.26 −0.46 −3.07 −1.44 −0.60 −1.61 0.05 086 Q086G 0.21 1.02 −1.18 −0.54 −0.42 −2.55 0.12 086 Q086H 1.34 1.23 −1.23 −0.61 −2.15 −1.84 0.10 086 Q086I 0.84 0.85 0.88 0.95 0.85 0.81 0.99 086 Q086K 0.64 0.66 0.88 0.84 0.71 0.81 1.42 086 Q086L 0.71 0.71 0.78 0.80 0.71 0.70 1.24 086 Q086N −4.91 −2.76 5.03 2.01 3.01 2.98 −0.02 086 Q086P 1.13 1.17 1.36 1.48 1.31 1.11 0.86 086 Q086R −7.06 24.56 −23.74 −11.36 −12.19 −55.94 0.00 086 Q086S 0.44 1.09 −1.75 −0.70 −0.40 −2.57 0.08 086 Q086T 0.94 0.91 0.90 0.99 0.78 0.91 1.03 086 Q086V 0.88 0.89 0.85 0.91 0.81 0.71 0.93 086 Q086W 0.78 0.74 0.81 0.85 0.80 0.69 1.24 086 Q086Y 0.72 0.74 0.94 0.91 0.77 0.82 1.13 089 Q089A 0.33 −0.78 0.99 0.42 −0.25 3.87 −0.12 089 Q089C −0.69 0.00 0.05 −0.23 −0.17 0.59 −0.11 089 Q089D 1.32 1.36 1.41 1.47 1.37 1.07 0.73 089 Q089E −0.37 −0.82 0.68 0.35 0.65 1.13 −0.12 089 Q089F 1.56 1.48 1.17 1.25 1.54 1.00 0.44 089 Q089G 1.13 1.03 1.21 1.15 1.10 1.02 0.93 089 Q089H 1.82 1.91 0.35 0.65 1.10 1.61 0.17 089 Q089I 1.16 1.01 1.04 1.10 1.15 0.90 0.77 089 Q089K 0.85 0.87 1.20 1.06 1.11 0.87 1.10 089 Q089L 0.95 0.94 0.97 0.98 0.72 0.77 0.86 089 Q089M 1.29 1.21 1.53 1.56 1.40 1.16 0.64 089 Q089N 1.40 1.30 1.64 1.67 1.56 1.26 0.71 089 Q089P −0.80 0.03 1.23 0.45 0.97 2.98 −0.11 089 Q089R 1.15 1.00 1.27 1.34 1.15 1.13 0.95 089 Q089T 11.41 −3.81 −9.90 −2.46 −5.57 −36.35 0.02 089 Q089V 1.15 0.96 1.24 1.23 1.20 0.84 0.90 089 Q089W 0.84 0.66 0.84 0.84 0.79 0.69 1.11 089 Q089Y 0.97 0.97 1.23 1.19 1.06 0.88 0.95 093 A093C 1.36 1.43 1.51 1.80 1.74 1.33 0.57 093 A093D 1.21 1.41 1.52 1.53 1.26 1.12 0.80 093 A093E 1.53 1.50 1.78 1.75 1.55 1.55 0.71 093 A093F 1.24 1.42 1.20 1.45 1.54 1.13 0.72 093 A093G 1.20 1.15 1.23 1.29 1.35 1.10 0.89 093 A093H 0.98 0.88 1.01 1.03 1.01 0.91 1.11 093 A093I 0.97 1.11 1.11 1.39 1.28 1.16 0.76 093 A093K 0.93 0.92 1.10 1.07 0.87 0.86 1.14 093 A093L 0.90 0.91 1.08 1.09 0.97 1.03 0.96 093 A093M 1.10 1.13 1.45 1.53 1.42 1.34 0.82 093 A093N 1.52 1.46 1.77 1.72 1.46 1.59 0.73 093 A093P −0.84 −0.82 0.20 −0.65 −0.46 −0.39 −0.09 093 A093Q 1.36 1.45 1.41 1.56 1.41 1.40 0.76 093 A093R 1.15 1.13 1.23 1.32 1.29 1.04 0.91 093 A093S 1.09 1.11 1.30 1.22 1.02 0.93 0.94 093 A093T 0.93 0.90 1.02 1.05 0.89 0.98 1.02 093 A093V 1.02 1.08 1.11 1.16 1.10 0.93 0.87 093 A093W 1.02 0.97 0.98 1.05 0.92 0.82 0.88 093 A093Y 0.93 0.91 1.17 1.22 1.12 1.12 0.83 115 W115D 0.97 1.05 0.95 1.04 1.15 1.05 0.91 115 W115E 0.88 0.90 0.91 0.96 0.69 0.88 1.09 115 W115F 0.87 0.92 0.79 0.85 0.95 0.73 1.11 115 W115G 0.81 0.81 0.93 0.89 0.84 1.10 1.28 115 W115K 0.67 0.64 0.75 0.74 0.73 0.62 1.44 115 W115L 0.58 0.58 0.67 0.65 0.62 0.69 1.63 115 W115N 0.87 0.94 1.13 1.07 1.08 0.98 1.02 115 W115P 1.12 1.12 1.18 1.20 1.22 1.29 0.87 115 W115Q 0.96 0.96 1.01 1.04 0.99 0.85 1.00 115 W115R 0.79 0.83 0.92 0.94 0.95 0.65 1.07 115 W115S 0.92 0.87 1.02 0.98 0.96 1.00 1.14 115 W115V 0.77 0.81 0.83 0.79 0.89 0.82 1.27 115 W115Y 0.56 0.63 0.74 0.73 0.77 0.69 1.41 117 D117A 1.29 1.21 1.20 1.32 1.83 1.39 0.45 117 D117E 1.40 1.34 1.30 1.21 1.65 1.33 0.65 117 D117G 1.10 1.02 1.08 1.08 1.19 1.33 0.78 117 D117H 0.92 0.82 0.84 0.89 1.21 1.08 0.87 117 D117I 0.98 0.70 0.65 0.79 1.49 1.26 0.50 117 D117K 0.68 0.57 0.93 0.89 0.82 0.86 1.30 117 D117L 0.81 0.77 0.94 0.86 1.09 0.86 0.64 117 D117M 1.21 1.13 1.23 1.32 1.44 0.97 0.53 117 D117N 1.30 1.26 1.63 1.48 1.48 1.23 0.71 117 D117P 1.07 1.04 1.27 1.17 1.29 1.10 0.88 117 D117Q 1.63 1.62 1.46 1.51 2.08 1.57 0.56 117 D117R 1.06 1.12 1.12 1.05 1.25 0.83 0.95 117 D117S 0.89 0.90 0.92 0.98 1.09 0.94 0.81 117 D117T 0.93 0.83 0.82 0.88 1.03 1.20 0.91 117 D117V 1.08 0.86 0.91 1.08 2.00 1.98 0.40 117 D117W 0.76 0.56 0.82 0.82 0.74 0.70 1.20 123 P123A 1.06 1.01 1.43 1.25 1.10 0.95 0.84 123 P123D 1.19 1.05 1.18 1.22 1.38 1.34 0.84 123 P123E 1.49 1.39 1.45 1.41 1.65 1.24 0.66 123 P123G 1.10 0.96 1.18 1.07 1.18 1.10 0.90 123 P123K 0.84 0.62 1.09 0.96 1.00 0.89 1.18 123 P123L 0.83 0.72 1.03 1.00 1.21 0.98 0.93 123 P123M 1.14 1.00 1.13 1.29 1.52 1.25 0.61 123 P123Q 1.25 1.15 1.19 1.35 1.55 1.30 0.68 123 P123R 1.02 0.95 1.28 1.28 1.12 1.11 0.96 123 P123S 1.07 0.84 0.88 0.92 1.17 0.97 0.96 123 P123T 1.00 0.89 0.83 0.90 1.13 0.83 0.97 123 P123V 0.83 0.79 0.97 1.08 1.23 0.99 0.83 124 S124A 1.33 1.45 1.31 1.39 1.27 1.34 0.79 124 S124C 1.28 1.32 0.93 1.15 1.54 1.21 0.77 124 S124D 1.19 1.20 1.14 1.24 1.35 1.31 0.88 124 S124E 1.15 1.22 1.16 1.19 1.24 1.11 0.93 124 S124F 1.12 1.19 1.05 1.16 1.05 0.98 1.01 124 S124G 0.92 0.81 0.79 0.84 1.14 0.80 1.08 124 S124H 0.91 0.98 0.97 1.02 1.12 1.11 0.98 124 S124I 0.93 0.92 0.92 0.91 0.99 1.05 1.10 124 S124K 0.89 0.89 0.95 0.90 0.92 0.77 1.24 124 S124L 0.70 0.73 0.74 0.62 0.74 0.61 1.36 124 S124N 1.16 1.18 1.00 1.15 1.17 0.96 0.87 124 S124P 1.08 0.97 1.06 1.25 1.43 1.02 0.71 124 S124Q 1.16 1.22 1.09 1.18 1.27 1.20 0.82 124 S124R 1.26 1.24 1.05 1.16 1.35 1.21 0.88 124 S124T 1.03 1.09 0.90 0.92 1.06 1.03 1.02 124 S124V 0.97 0.96 0.81 0.86 0.98 1.00 1.01 124 S124Y 0.87 0.88 0.74 0.83 0.86 0.75 1.13 125 D125A 0.75 0.72 1.11 1.21 1.17 1.04 0.82 125 D125E 1.02 1.00 0.92 1.01 0.96 0.77 0.94 125 D125G 0.51 0.48 0.91 0.90 0.46 0.77 1.15 125 D125K 0.37 0.34 0.65 0.80 0.79 0.68 1.23 125 D125M 1.08 1.06 1.02 1.11 1.11 0.98 0.93 125 D125Q 0.87 0.74 0.92 1.03 0.85 0.81 0.94 125 D125R 0.43 0.43 0.69 0.92 0.74 0.61 1.06 125 D125S 0.67 0.57 0.96 1.03 0.80 0.82 1.12 125 D125T 0.91 0.92 0.78 0.80 0.78 0.59 1.17 125 D125V 0.38 0.36 0.67 0.81 0.78 1.01 1.17 127 N127A 0.80 0.88 1.41 1.37 1.42 1.34 0.69 127 N127C 1.25 1.40 1.26 1.39 1.48 1.45 0.69 127 N127D 1.21 1.24 1.24 1.31 1.19 1.41 0.78 127 N127F 0.98 0.84 1.27 1.23 1.09 0.90 0.82 127 N127G 0.86 0.71 1.05 1.04 1.03 1.05 0.93 127 N127H 0.70 0.63 0.83 0.89 0.86 0.81 1.02 127 N127K 0.51 0.47 0.77 0.86 0.95 1.01 1.03 127 N127L 0.71 0.67 0.99 1.04 1.12 1.01 0.93 127 N127M 1.00 1.15 1.47 1.46 1.35 1.23 0.73 127 N127P 1.10 1.04 1.30 1.39 1.16 1.21 0.65 127 N127Q 1.04 1.01 1.42 1.47 1.33 1.00 0.71 127 N127R 0.88 0.87 1.04 1.10 1.14 0.89 0.96 127 N127S 0.87 0.71 1.08 1.06 1.04 0.91 0.90 127 N127T 0.78 0.68 0.79 0.79 0.93 0.75 1.08 127 N127V 0.75 0.78 1.34 1.27 1.11 0.97 0.75 127 N127W 0.79 0.73 0.76 0.80 0.84 0.82 1.16 127 N127Y 1.16 1.12 1.23 1.15 1.21 1.14 0.82 130 I130A 1.15 1.06 1.61 1.47 1.25 1.13 0.82 130 I130G 0.81 0.71 1.10 1.12 1.25 1.11 0.91 130 I130H 0.58 0.51 0.83 0.87 0.92 0.79 1.18 130 I130K 0.73 0.64 1.06 0.96 0.94 0.84 1.14 130 I130L 0.80 0.77 1.08 1.04 0.99 0.92 0.96 130 I130M 1.45 1.37 1.42 1.44 1.33 1.19 0.77 130 I130N 1.08 0.95 1.48 1.46 1.11 1.01 0.78 130 I130P 0.29 0.41 0.91 1.29 1.33 1.36 0.76 130 I130Q −0.18 −1.25 1.18 0.52 −0.19 0.03 −0.12 130 I130R 1.09 0.91 1.30 1.34 0.91 1.00 0.85 130 I130S 1.02 0.81 1.02 1.06 1.07 0.84 0.97 130 I130T 0.97 0.99 1.09 1.04 0.97 0.99 0.98 130 I130V 1.12 1.21 1.12 1.12 0.94 0.96 0.83 130 I130W 0.85 0.69 0.95 1.01 1.11 0.89 0.89 132 G132A 1.52 1.66 1.32 1.47 1.60 1.39 0.68 132 G132C 1.38 1.47 1.26 1.28 1.10 0.87 0.79 132 G132D 1.84 1.78 1.18 1.50 1.56 1.56 0.51 132 G132E 1.17 1.33 1.11 1.21 0.90 0.86 0.86 132 G132F 1.20 1.21 1.03 1.18 1.03 1.03 0.91 132 G132H 0.98 1.03 0.93 0.97 1.04 0.99 1.02 132 G132L 0.98 1.03 0.76 0.84 0.88 0.85 0.95 132 G132M 1.81 1.76 1.51 1.61 1.81 1.73 0.61 132 G132N 1.18 1.23 1.11 1.27 1.15 1.08 0.82 132 G132P 1.51 1.59 1.28 1.40 1.26 1.34 0.75 132 G132R 1.35 1.32 0.97 1.12 1.02 1.06 0.87 132 G132S 1.32 1.34 1.08 1.22 1.15 0.96 0.77 132 G132T 1.09 1.05 0.87 0.96 0.94 0.98 1.03 132 G132V 0.99 1.09 0.96 1.02 0.95 1.03 0.98 132 G132W 1.17 1.13 0.95 1.07 1.02 0.84 0.91 132 G132Y 0.93 0.87 0.81 0.86 0.80 0.81 1.15 135 Q135A 0.95 0.99 1.00 1.20 1.13 0.99 0.84 135 Q135F 0.93 1.05 0.91 1.03 1.04 1.39 0.94 135 Q135G 0.77 0.80 0.75 0.84 0.97 1.05 1.08 135 Q135K 0.59 0.69 0.63 0.71 0.70 0.74 1.27 135 Q135L 0.40 0.22 −0.66 −0.33 0.38 −0.14 0.19 135 Q135M 0.91 1.03 1.01 1.14 0.90 1.01 0.86 135 Q135P 1.12 1.16 0.96 1.07 0.85 1.16 0.87 135 Q135R 0.92 1.01 0.95 1.00 0.95 1.14 1.08 135 Q135S 0.93 0.93 0.80 0.95 0.91 0.87 0.94 135 Q135T 0.84 0.83 0.73 0.80 0.79 0.83 1.19 135 Q135V 0.92 0.84 0.66 0.75 0.98 1.23 0.76 135 Q135Y 0.75 0.75 0.69 0.79 0.75 0.91 1.16 145 P145A 1.15 1.17 1.44 1.50 1.30 1.29 0.71 145 P145D 1.35 1.61 1.15 1.33 1.45 1.48 0.57 145 P145E 1.55 1.48 1.26 1.45 1.34 1.65 0.63 145 P145F 1.34 1.32 1.02 1.20 1.12 1.32 0.74 145 P145H 0.98 0.88 0.81 0.99 1.13 1.03 0.90 145 P145I 1.20 1.06 0.84 0.98 1.10 1.20 0.85 145 P145K 0.90 0.89 0.88 1.05 1.15 1.02 0.88 145 P145L 1.03 0.90 0.81 1.01 0.97 1.23 0.85 145 P145N 1.19 1.43 1.41 1.58 1.49 1.48 0.61 145 P145R 1.22 1.11 1.10 1.29 1.14 1.11 0.86 145 P145S 1.12 1.04 1.03 1.10 1.08 1.12 0.85 145 P145T 1.15 0.99 0.91 1.00 0.92 0.81 0.89 145 P145V 1.09 1.18 1.08 1.19 1.24 1.25 0.73 145 P145Y 1.14 1.12 0.89 1.15 1.08 1.16 0.75 146 G146A 1.18 1.00 1.34 1.41 1.19 1.05 0.77 146 G146C 2.03 1.89 1.71 1.89 2.19 1.52 0.40 146 G146D 1.32 1.35 1.45 1.52 1.50 1.17 0.70 146 G146E 1.43 1.43 1.63 1.62 1.38 1.33 0.82 146 G146H 1.00 0.99 0.77 0.88 1.01 0.93 1.06 146 G146K 0.94 0.84 1.06 1.07 1.05 1.10 1.08 146 G146L 0.99 0.80 0.85 ND 0.97 0.74 0.97 146 G146P 1.33 1.24 1.30 1.36 1.43 1.31 0.76 146 G146R 1.21 1.07 1.12 1.30 1.21 1.41 0.85 146 G146S 1.18 1.13 1.02 1.04 0.97 0.94 0.91 146 G146T 0.91 0.83 0.98 1.00 0.97 0.78 1.13 146 G146V 1.19 1.04 1.15 1.21 1.22 1.16 0.79 146 G146W 0.94 0.88 0.83 0.91 0.80 1.01 0.93 148 G148A 1.20 1.28 1.37 1.49 1.22 1.10 0.71 148 G148C 1.09 0.94 1.25 1.28 1.17 1.19 0.84 148 G148D −1.07 −0.40 0.77 0.36 0.81 1.35 −0.11 148 G148E 1.17 1.19 1.27 1.27 1.37 1.02 0.89 148 G148F 1.08 1.07 1.13 1.16 1.03 1.27 0.97 148 G148H 0.89 0.90 1.00 0.97 0.91 0.66 1.13 148 G148L 0.88 0.83 0.89 0.90 0.94 0.91 1.16 148 G148N 1.00 0.98 1.20 1.17 1.13 1.18 1.01 148 G148P 1.30 1.33 1.34 1.45 1.35 1.09 0.76 148 G148Q −1.69 −1.32 1.69 0.84 −0.01 0.58 −0.07 148 G148R 1.27 1.20 1.12 1.24 1.14 0.71 0.85 148 G148S 0.96 0.97 0.92 0.96 0.84 1.05 1.03 148 G148T 1.35 1.20 0.70 0.86 1.35 0.90 0.49 148 G148V 0.96 0.95 0.95 1.02 1.04 0.69 1.03 148 G148W 1.00 0.88 0.90 0.96 0.89 0.83 1.00 148 G148Y 0.82 0.74 0.86 0.93 0.91 0.87 1.14 153 S153A 1.14 1.15 1.09 1.23 1.21 1.09 0.86 153 S153C 1.70 1.78 1.37 1.54 1.51 1.61 0.53 153 S153D 1.34 1.25 0.99 1.11 1.18 1.02 0.88 153 S153E 1.76 1.18 1.20 1.34 1.54 1.36 0.61 153 S153F 1.42 1.35 1.07 1.18 1.22 1.36 0.80 153 S153G 1.35 1.16 0.97 1.03 1.08 1.25 0.88 153 S153H 0.98 0.91 0.72 0.77 0.92 1.00 1.05 153 S153I 1.31 1.09 0.95 1.05 1.28 1.35 0.74 153 S153K 0.79 0.82 0.80 0.87 1.02 1.00 1.08 153 S153L 1.11 0.96 0.92 0.98 1.03 1.26 0.92 153 S153N 1.29 1.38 1.38 1.43 1.20 1.75 0.76 153 S153P 1.09 1.19 1.21 1.28 1.15 1.15 1.00 153 S153Q 1.14 1.22 0.95 1.21 1.39 1.47 0.73 153 S153R 1.29 1.16 1.00 1.12 1.18 1.16 0.95 153 S153T 0.93 1.02 0.82 0.89 0.88 0.97 1.01 153 S153V 1.30 1.16 0.97 1.07 1.08 1.26 0.77 153 S153W 0.94 0.95 0.75 0.86 0.85 1.21 0.90 153 S153Y 1.15 1.03 0.96 1.00 1.03 1.13 0.86 159 Y159A 1.17 1.17 1.42 1.52 1.50 1.70 0.74 159 Y159C 1.46 1.19 1.80 1.73 1.10 1.66 0.66 159 Y159D 1.08 1.21 1.89 1.88 1.63 2.01 0.78 159 Y159E 1.23 1.25 1.34 1.50 1.31 1.66 0.73 159 Y159F 1.11 1.10 1.03 1.10 0.96 1.17 0.97 159 Y159G 0.92 0.79 0.95 1.02 1.22 1.44 0.94 159 Y159H 0.97 0.86 0.97 1.05 1.07 1.17 1.01 159 Y159K 1.03 0.78 1.20 1.21 0.96 1.71 0.90 159 Y159L 0.77 0.61 0.95 1.00 1.24 1.41 0.98 159 Y159N 0.97 0.94 1.46 1.56 1.40 1.85 0.78 159 Y159R 1.10 0.86 1.19 1.40 1.39 1.75 0.80 159 Y159S 0.97 0.82 1.01 1.09 1.25 1.44 0.94 159 Y159T 0.93 0.93 1.18 1.20 1.26 1.74 0.87 159 Y159V 1.09 0.95 0.96 1.07 1.12 1.48 0.87 159 Y159W 1.08 1.12 0.98 1.08 0.73 0.96 0.88 166 W166C 0.57 0.71 0.61 0.60 0.90 1.03 0.80 166 W166E 0.58 0.78 0.94 0.74 1.09 1.12 0.97 166 W166F 0.75 0.82 0.73 0.81 0.82 1.06 1.07 166 W166G 0.65 0.57 0.42 0.50 0.84 0.93 1.18 166 W166H 0.59 0.64 0.57 0.67 0.90 0.92 1.26 166 W166I 0.56 0.65 0.85 0.79 0.65 1.16 1.13 166 W166K 0.47 0.49 0.96 0.81 0.60 1.09 1.49 166 W166L 0.45 0.54 0.79 0.64 0.58 0.91 1.46 166 W166M 0.70 0.83 0.96 0.82 0.89 1.24 1.00 166 W166P 0.16 0.30 −0.15 −0.02 1.19 1.09 0.69 166 W166R 0.67 0.86 1.40 1.17 0.83 0.76 0.94 166 W166S 0.61 0.64 0.63 0.57 0.90 0.95 1.14 166 W166T 0.60 0.73 0.92 0.76 0.70 0.33 1.15 166 W166V 0.51 0.61 0.88 0.75 0.49 0.98 1.10 166 W166Y 0.65 0.70 0.73 0.78 0.81 0.96 1.16 169 S169A 1.10 1.14 1.17 1.30 1.17 1.35 0.85 169 S169C 1.05 1.37 1.33 1.43 1.54 1.47 0.74 169 S169D 0.91 1.17 1.21 1.26 1.36 1.42 0.71 169 S169E 1.24 1.36 1.37 1.59 1.65 1.81 0.67 169 S169F 1.04 1.33 1.01 1.13 1.15 1.36 0.74 169 S169G 1.05 0.90 0.97 0.99 0.99 1.22 0.97 169 S169I 0.84 0.82 0.84 1.02 1.18 1.00 0.93 169 S169K 0.81 0.85 0.78 0.94 0.88 1.03 1.00 169 S169L 0.82 0.65 0.83 0.95 0.99 1.00 1.09 169 S169M 0.98 0.71 1.05 1.17 0.98 1.17 0.92 169 S169N 0.88 1.09 1.28 1.35 1.43 1.41 0.76 169 S169P 0.55 0.85 0.74 1.10 1.25 0.96 0.78 169 S169Q 1.08 1.18 1.27 1.43 1.43 1.54 0.75 169 S169R 0.95 0.88 1.01 1.13 1.08 1.26 0.96 169 S169T 0.78 0.75 0.87 0.96 0.92 0.79 1.07 169 S169V 0.78 0.88 0.78 1.01 1.10 1.00 0.94 169 S169Y 0.91 0.87 1.00 1.11 0.97 1.10 0.93 171 K171C 1.20 1.53 1.27 1.46 1.71 1.56 0.54 171 K171D 1.22 1.28 1.11 1.32 1.03 1.49 0.75 171 K171E 1.18 1.46 1.26 1.33 1.61 1.36 0.65 171 K171G 1.03 1.06 0.87 0.91 1.08 1.29 0.80 171 K171H 0.90 0.76 0.82 0.90 0.93 0.94 1.20 171 K171L 0.70 0.76 0.84 0.88 0.88 0.86 1.05 171 K171M 1.11 1.32 1.25 1.34 1.43 1.08 0.67 171 K171P 0.99 1.09 1.19 1.31 1.36 1.32 0.73 171 K171Q 1.08 1.15 1.05 1.26 1.66 1.37 0.67 171 K171R 1.01 0.96 0.96 1.08 1.10 1.42 0.92 171 K171S 1.12 0.97 0.86 0.96 0.90 1.17 0.93 171 K171T 1.00 0.88 0.87 0.93 0.96 1.03 1.09 171 K171V 0.87 0.92 0.81 0.92 1.13 0.97 0.89 171 K171W 0.74 0.74 0.59 0.82 0.88 0.86 1.03 171 K171Y 0.86 0.73 0.70 0.90 0.96 1.06 0.92 179 R179A 0.88 0.88 1.28 1.47 1.62 1.55 0.83 179 R179G 0.53 0.56 0.93 1.05 1.02 1.09 1.05 179 R179H 0.82 0.78 1.07 1.19 1.25 1.32 1.00 179 R179L 0.71 0.62 0.79 0.93 1.00 1.28 0.99 179 R179M 0.81 1.14 1.24 1.51 1.58 1.52 0.66 179 R179P 0.33 0.50 1.14 1.33 1.42 1.61 0.79 179 R179Q 1.07 0.98 1.03 1.27 1.31 1.39 0.82 179 R179S 0.86 0.62 0.94 1.12 1.11 1.21 1.02 179 R179T 0.97 0.78 1.00 1.16 1.31 1.32 0.94 179 R179V 0.90 0.89 0.84 1.03 1.26 1.12 0.91 179 R179W 0.81 0.70 0.92 1.19 1.17 1.71 0.87 179 R179Y 0.64 0.49 0.81 0.95 0.89 1.22 1.06 180 G180A 0.82 0.75 1.37 0.96 1.25 1.45 0.76 180 G180C 0.43 0.46 1.29 0.82 1.35 1.56 0.73 180 G180D 0.50 0.54 1.39 0.99 1.37 1.36 0.81 180 G180F 0.32 0.32 1.19 1.03 0.90 1.75 0.72 180 G180H 0.38 0.36 0.95 0.80 0.89 1.00 1.12 180 G180I 0.21 0.21 0.87 0.73 1.17 1.09 0.84 180 G180K 0.13 0.16 0.77 ND 0.99 1.02 1.26 180 G180L 0.22 0.25 1.02 0.72 0.81 0.94 1.38 180 G180N 0.46 0.52 1.41 1.13 1.24 0.89 0.81 180 G180P 0.42 0.45 1.39 ND 1.46 1.74 0.75 180 G180R 1.27 0.83 ND ND −0.82 −12.79 −0.03 180 G180S 0.82 0.70 0.98 0.79 0.90 0.92 0.96 180 G180T 0.46 0.37 0.91 0.76 1.02 0.77 1.07 180 G180V 0.25 0.18 0.89 ND 1.01 0.98 0.96 180 G180Y 0.29 0.34 0.91 0.75 1.03 1.11 1.03 181 I181A 1.16 1.15 1.60 1.19 1.45 1.48 0.78 181 I181C 1.18 1.21 1.79 ND 1.10 1.35 0.68 181 I181D 1.19 1.29 1.61 1.22 1.64 1.19 0.74 181 I181E 1.44 1.47 1.54 1.27 1.48 1.55 0.72 181 I181F 1.11 1.04 1.20 0.92 1.24 0.84 0.87 181 I181G 0.69 0.59 1.20 1.02 1.15 1.12 0.94 181 I181H 0.88 0.73 1.13 ND 0.95 0.95 0.98 181 I181K 0.58 0.43 1.15 ND 0.96 0.87 1.04 181 I181L 0.76 0.75 1.09 ND 0.91 0.78 1.01 181 I181P 1.04 1.07 1.35 ND 1.30 1.34 0.85 181 I181R 0.49 0.47 0.78 0.87 1.43 1.64 0.87 181 I181S 0.93 0.83 1.09 0.80 1.02 1.15 0.97 181 I181T 0.80 0.75 1.02 0.85 0.94 1.13 1.09 181 I181V 1.20 1.04 1.39 1.12 1.18 1.16 0.81 181 I181Y 0.83 0.69 1.06 0.89 0.94 0.87 0.95 182 G182A 0.82 0.87 1.36 1.14 1.24 1.11 0.72 182 G182C 1.18 1.14 1.73 1.58 1.96 1.42 0.45 182 G182D 0.79 0.71 1.40 1.17 1.40 1.56 0.77 182 G182E 0.68 0.64 1.45 1.26 1.73 1.43 0.66 182 G182F 1.30 1.21 1.10 1.06 1.50 1.46 0.46 182 G182H 0.64 0.53 1.01 ND 1.13 1.22 1.01 182 G182K −0.04 −0.01 −0.15 ND 0.07 −0.08 0.91 182 G182L 0.53 0.48 1.12 ND 1.26 0.86 0.70 182 G182P 0.61 0.47 1.45 1.23 1.57 1.40 0.73 182 G182R 0.77 0.69 1.32 0.99 1.30 1.56 0.81 182 G182S 0.68 0.57 0.99 0.87 1.16 1.10 0.85 182 G182T 0.49 0.49 1.00 0.89 1.22 1.12 0.84 182 G182V 0.61 0.46 1.26 1.12 1.38 1.18 0.66 182 G182Y 0.61 0.48 0.85 0.88 1.00 0.96 0.84 183 K183A 0.20 0.17 1.19 1.20 1.58 1.99 0.66 183 K183C 0.18 0.19 1.35 1.24 1.38 2.02 0.62 183 K183E 0.14 0.02 1.20 1.10 1.37 1.55 0.72 183 K183F 0.15 −0.05 1.11 1.08 1.61 2.04 0.59 183 K183G 0.15 0.13 0.96 ND 1.24 1.35 0.94 183 K183H 0.27 0.15 0.93 0.84 1.31 1.26 0.86 183 K183L 0.05 0.10 0.91 0.93 1.15 1.44 0.86 183 K183M 0.14 0.18 1.42 1.40 1.60 2.40 0.58 183 K183P 0.03 0.17 1.27 1.06 1.73 1.64 0.58 183 K183Q 0.12 0.14 1.30 1.21 1.51 2.11 0.71 183 K183R 0.20 0.11 1.03 1.00 1.13 2.07 0.82 183 K183S 0.15 0.18 0.92 0.83 1.18 1.33 0.93 183 K183T 0.20 0.01 1.10 1.11 1.16 1.71 0.80 183 K183V 0.10 0.07 0.82 0.89 1.38 1.73 0.62 183 K183W 0.07 0.13 0.66 0.77 1.23 1.09 0.70 183 K183Y 0.08 0.14 0.66 0.73 1.10 1.32 0.88 187 W187A 0.45 0.36 1.67 1.77 1.21 1.02 0.90 187 W187C 0.48 0.40 1.23 1.49 1.19 1.13 0.88 187 W187E 0.62 0.51 1.18 1.22 1.14 1.08 1.04 187 W187G 0.16 0.13 0.57 0.86 0.86 0.73 1.13 187 W187I 0.70 0.57 0.83 0.86 0.78 0.82 1.29 187 W187K 0.16 0.16 0.85 0.90 0.71 0.64 1.72 187 W187L 0.69 0.69 0.72 0.78 0.75 0.66 1.29 187 W187N 0.53 0.62 1.29 1.41 1.10 1.08 0.91 187 W187P 0.17 0.14 0.29 1.65 1.19 1.15 0.85 187 W187Q 0.41 0.35 1.27 1.30 1.09 0.92 1.03 187 W187R 0.29 0.28 0.96 1.19 0.92 0.92 1.08 187 W187S 0.40 0.28 1.01 1.07 0.86 0.76 1.25 187 W187V 0.20 −0.60 −0.25 0.12 0.26 0.77 0.20 194 G194A 1.33 1.26 1.40 1.36 1.55 1.18 0.68 194 G194E 1.28 1.12 1.13 1.10 1.37 1.25 0.79 194 G194H 1.09 1.02 0.91 0.92 1.10 0.72 0.97 194 G194K 0.77 0.75 0.80 0.80 1.01 0.68 1.18 194 G194L 0.74 0.64 0.81 0.85 0.97 0.78 1.01 194 G194M 1.38 1.33 1.65 1.48 1.40 1.19 0.69 194 G194P 0.04 0.01 0.00 2.12 1.14 0.37 0.78 194 G194R 1.04 0.96 0.98 1.05 1.08 0.86 1.01 194 G194S 1.08 1.02 1.14 1.09 0.94 0.70 1.03 194 G194T 0.93 0.77 0.87 0.92 1.05 0.71 1.07 194 G194V 1.26 1.08 0.97 1.01 1.14 0.80 0.84 194 G194W 0.61 0.51 0.60 0.62 0.75 0.48 1.31 209 P209A 1.22 1.30 1.67 1.68 1.52 1.20 0.68 209 P209C 1.24 1.20 1.01 1.00 1.37 1.29 0.58 209 P209D 1.35 1.33 1.41 1.57 1.39 1.30 0.76 209 P209E 1.34 1.25 1.31 1.47 1.34 1.35 0.82 209 P209F 1.23 1.36 1.22 1.48 1.51 1.50 0.62 209 P209G 1.09 0.92 1.10 1.09 0.98 0.91 1.01 209 P209H 1.02 0.91 1.20 1.15 0.97 1.00 1.00 209 P209I 0.94 0.85 1.01 1.03 0.91 0.84 0.95 209 P209K 1.24 0.95 1.22 1.23 0.99 1.03 0.98 209 P209L 0.83 0.92 1.00 1.06 1.02 1.02 0.87 209 P209M 1.34 1.34 1.48 1.61 1.81 1.18 0.65 209 P209N 1.65 2.42 0.99 1.11 2.77 2.36 0.21 209 P209Q −0.73 −0.45 ND ND −0.20 0.55 −0.12 209 P209R 1.38 1.03 1.41 1.45 1.08 1.22 0.91 209 P209S 1.14 1.02 1.13 1.21 1.02 0.86 0.89 209 P209T 1.12 1.10 1.37 1.35 1.35 0.97 0.84 209 P209V 0.98 0.83 1.10 0.93 1.16 1.04 0.82 209 P209W 0.99 1.06 1.03 1.10 1.16 0.86 0.80 209 P209Y 0.64 0.73 0.53 0.76 0.96 1.10 0.82 224 N224A 0.65 1.07 1.11 1.13 1.47 1.19 0.81 224 N224C 0.91 1.07 1.05 1.08 1.22 1.00 0.82 224 N224D 0.81 1.10 0.96 1.08 0.85 0.75 0.82 224 N224E 0.93 1.78 0.68 0.91 1.68 1.83 0.31 224 N224F 0.79 1.11 0.81 0.93 1.33 1.31 0.67 224 N224G 0.72 0.80 0.82 0.87 0.72 0.97 1.26 224 N224H 0.60 0.80 0.74 0.84 0.66 0.83 1.20 224 N224I 0.71 0.95 0.85 0.99 0.95 0.97 0.86 224 N224K 0.56 0.64 0.78 0.81 0.58 0.78 1.42 224 N224L 0.55 0.72 0.69 0.75 0.89 0.73 1.11 224 N224M 0.84 1.07 1.07 1.13 1.22 1.27 0.73 224 N224P 1.21 1.26 0.87 1.03 1.04 1.19 0.83 224 N224Q 0.97 1.03 0.98 0.98 1.12 1.07 0.90 224 N224R 0.89 1.22 0.65 0.76 1.15 1.34 0.53 224 N224S 0.91 0.84 0.88 0.93 0.78 0.82 1.11 224 N224T 0.90 0.86 0.93 0.95 0.78 0.99 1.17 224 N224V 0.83 0.88 0.78 0.89 0.92 0.89 0.92 224 N224W 0.82 0.87 0.70 0.80 0.79 0.88 1.02 224 N224Y 0.92 1.03 0.81 0.89 0.87 0.77 0.93 242 S242A 1.44 1.56 1.59 1.45 1.19 1.10 0.72 242 S242C 1.73 1.99 1.84 1.93 1.97 1.06 0.46 242 S242D 1.36 1.51 1.45 1.43 1.29 1.11 0.81 242 S242G 1.24 1.20 1.29 1.22 1.12 0.92 0.93 242 S242I 1.36 1.12 1.14 1.24 1.43 1.34 0.55 242 S242K 0.80 0.70 0.92 0.92 0.99 0.86 1.26 242 S242L 0.77 0.75 1.26 1.08 1.06 0.99 0.93 242 S242M 1.56 1.70 1.48 1.78 1.54 1.10 0.55 242 S242Q 1.44 1.62 1.28 1.34 1.34 1.14 0.84 242 S242R 0.98 0.82 1.14 1.15 1.02 1.07 1.05 242 S242T 1.06 0.93 0.90 0.91 1.08 0.71 0.99 242 S242V 1.19 1.00 0.90 1.00 1.28 0.94 0.65 245 P245A 1.53 1.58 1.74 1.69 1.46 1.09 0.68 245 P245C 1.18 1.01 1.45 1.43 1.25 1.07 0.63 245 P245D 1.52 1.36 1.65 1.61 1.45 1.53 0.77 245 P245E 1.39 1.24 1.28 1.40 1.24 0.80 0.95 245 P245F 1.28 1.35 1.13 1.15 1.23 0.93 0.77 245 P245H 1.11 0.94 1.16 1.06 1.03 0.75 1.09 245 P245I 0.81 0.61 1.05 0.96 0.93 0.90 0.97 245 P245L 0.82 0.71 0.92 0.92 1.05 0.90 0.98 245 P245M 1.18 1.20 1.76 1.74 1.60 1.27 0.67 245 P245N 1.70 1.40 1.62 1.60 1.40 1.42 0.79 245 P245Q 1.49 1.22 1.61 1.52 1.26 0.99 0.96 245 P245R 1.37 1.23 1.19 1.26 1.47 1.08 0.88 245 P245S 1.16 1.01 1.20 1.21 1.07 0.88 1.07 245 P245T 1.31 0.99 1.26 1.23 1.13 0.97 0.92 245 P245V 1.03 0.95 0.97 1.00 0.94 0.75 1.00 245 P245Y 0.89 0.87 1.09 0.99 0.88 0.65 1.15 256 G256A 1.13 1.22 1.07 1.22 1.09 1.29 0.70 256 G256C 0.94 1.02 0.83 0.95 0.92 1.05 0.64 256 G256D 0.98 1.00 1.18 1.17 1.01 1.15 0.95 256 G256E 0.84 0.87 0.81 0.86 0.71 1.24 1.04 256 G256H 0.71 0.74 0.74 0.77 0.83 0.70 1.18 256 G256I 0.75 0.93 0.43 0.54 0.96 0.78 0.71 256 G256K 0.58 0.60 0.56 0.65 0.68 0.84 1.38 256 G256L 0.60 0.68 0.56 0.66 0.76 0.86 0.91 256 G256M 1.15 1.07 0.97 1.13 1.14 1.24 0.67 256 G256N 0.94 0.97 1.18 1.23 1.05 1.01 0.98 256 G256P 1.50 1.45 0.71 0.89 1.51 1.67 0.46 256 G256R 1.02 0.96 0.86 0.96 1.08 0.32 0.91 256 G256S 0.96 0.96 0.79 0.87 0.82 0.98 0.95 256 G256T 0.90 0.93 0.62 0.67 0.87 0.80 0.96 256 G256V 0.89 0.84 0.77 0.77 1.07 1.11 0.72 256 G256W 0.74 0.77 0.53 0.60 0.78 0.84 0.81 269 D269A 1.71 1.92 1.19 1.67 1.38 2.24 0.23 269 D269C 1.67 1.68 1.02 1.35 1.95 2.39 0.28 269 D269F −8.17 −9.85 2.60 1.57 −14.68 −4.97 0.02 269 D269G 1.18 1.30 0.68 0.96 1.59 1.43 0.37 269 D269H 0.88 0.93 0.86 1.01 1.21 1.07 0.56 269 D269I −4.21 −1.61 −2.68 −0.84 1.26 3.06 0.03 269 D269K 0.61 0.49 0.73 0.93 0.98 1.08 0.68 269 D269M 2.30 4.29 1.94 2.82 6.32 9.02 0.02 269 D269N 1.20 1.07 1.39 1.58 1.07 1.36 0.68 269 D269P 1.10 1.31 0.98 1.54 2.14 2.17 0.19 269 D269Q 1.53 1.50 1.31 1.60 1.53 1.51 0.44 269 D269R 1.11 1.09 0.80 1.25 1.59 1.26 0.39 269 D269S 0.82 0.86 0.93 0.96 0.89 0.91 1.07 269 D269T 0.76 0.98 0.78 0.96 1.43 1.43 0.30 269 D269Y 1.05 0.91 0.73 1.06 1.46 1.47 0.20 271 N271A 1.26 1.33 1.32 1.38 1.07 1.21 0.79 271 N271D 1.25 1.29 1.25 1.37 1.21 1.35 0.80 271 N271F 1.75 1.69 1.17 1.33 1.37 1.75 0.64 271 N271H 0.79 0.76 0.84 0.80 0.89 0.91 1.10 271 N271I 1.19 1.10 1.12 1.21 1.16 1.42 0.67 271 N271K 0.79 0.75 0.92 0.93 0.94 1.20 1.15 271 N271L 0.98 0.95 1.24 1.07 0.93 0.93 0.81 271 N271M 1.25 1.29 1.40 1.44 1.16 1.59 0.68 271 N271P 1.42 1.54 1.12 1.23 1.61 1.59 0.40 271 N271S 0.99 1.03 0.96 1.04 1.02 1.10 0.97 271 N271T 0.91 0.89 1.00 0.99 0.90 0.83 0.98 271 N271V 0.70 0.88 0.80 1.01 1.07 1.08 0.76 271 N271W 0.99 0.93 0.83 0.96 1.02 0.69 0.80 271 N271Y 0.95 0.87 0.86 0.96 0.84 0.94 0.78 278 T278A 1.17 1.21 1.25 1.41 1.11 1.35 0.84 278 T278E 0.92 0.80 0.99 1.13 1.06 1.35 0.88 278 T278G −1.12 0.26 −4.08 −1.79 3.31 1.75 0.04 278 T278H 0.89 0.89 0.92 1.02 0.94 1.03 1.00 278 T278I 0.89 0.87 0.81 0.86 0.82 0.89 1.10 278 T278K 0.84 0.75 0.91 0.96 0.82 0.91 1.08 278 T278L 0.82 0.80 1.02 0.99 0.82 0.93 0.96 278 T278M 1.26 1.35 1.27 1.47 1.32 1.35 0.70 278 T278N 1.22 1.19 1.17 1.30 1.00 1.35 0.86 278 T278P 1.78 1.71 1.06 1.79 2.30 3.46 0.19 278 T278R 1.13 1.07 1.00 1.24 1.10 1.47 0.83 278 T278S 0.91 0.92 0.86 1.00 0.99 1.01 1.06 278 T278W 0.92 0.99 0.84 1.00 1.04 1.08 0.85 278 T278Y 0.76 0.84 0.79 0.90 0.72 0.87 1.04 281 N281A 1.06 1.16 1.13 1.25 1.06 1.07 0.82 281 N281D 1.08 1.33 1.09 1.22 1.41 1.29 0.61 281 N281G 0.78 0.79 0.93 0.93 0.82 0.90 1.21 281 N281H 0.76 0.82 0.83 0.87 0.73 0.63 1.10 281 N281I 0.84 0.81 0.79 0.84 0.79 0.78 1.17 281 N281L 0.66 0.72 0.77 0.78 0.58 0.55 1.43 281 N281M 1.15 1.12 0.98 1.12 0.93 1.08 0.84 281 N281P 1.15 1.18 1.09 1.18 1.05 0.89 0.85 281 N281Q 1.22 1.33 1.00 1.13 1.10 1.16 0.62 281 N281R 1.03 1.05 0.98 1.02 0.95 0.97 0.96 281 N281S 0.90 0.91 0.88 0.89 0.72 1.01 1.09 281 N281T −0.21 1.17 −0.65 −0.27 −0.63 −0.72 0.14 281 N281V 0.85 0.88 0.80 0.87 0.76 1.07 0.86 281 N281Y 0.79 0.84 0.75 0.80 0.59 0.73 1.18 302 G302C 1.40 1.39 1.42 1.50 1.34 1.39 0.66 302 G302D 1.10 1.14 1.13 1.17 1.14 0.96 0.88 302 G302E 1.25 1.34 1.43 1.42 1.38 1.28 0.75 302 G302F 1.26 1.51 1.02 1.22 1.42 1.77 0.50 302 G302H 0.95 0.94 0.94 0.92 0.80 0.84 1.09 302 G302I 1.34 1.31 1.13 1.25 1.63 1.52 0.67 302 G302L 0.84 0.86 0.92 0.98 0.78 0.86 0.98 302 G302M 1.31 1.35 1.35 1.51 1.31 1.52 0.69 302 G302N 1.38 1.39 1.48 1.48 1.17 1.18 0.70 302 G302P 1.24 1.29 1.32 1.43 1.23 1.03 0.59 302 G302R 1.13 1.19 1.06 1.14 0.99 0.94 0.89 302 G302S 1.05 1.11 0.91 0.96 0.87 0.99 1.01 302 G302T 0.89 0.92 0.94 0.95 0.77 0.70 1.05 302 G302V 1.08 1.16 1.09 1.15 0.76 0.96 0.88 302 G302W 0.79 0.75 0.85 0.83 0.80 0.75 1.30 302 G302Y 0.91 0.98 0.91 0.99 0.75 0.92 0.93 304 A304D 1.19 1.30 1.42 1.44 1.16 1.22 0.75 304 A304E 1.70 1.57 1.41 1.47 1.45 1.45 0.69 304 A304F 1.14 1.14 1.27 1.18 0.86 0.87 1.02 304 A304H 0.85 0.85 0.77 0.83 0.81 0.68 1.15 304 A304L 0.92 0.95 0.91 0.96 0.80 0.74 1.00 304 A304M 1.18 1.21 1.45 1.49 0.98 1.18 0.72 304 A304N 1.42 1.41 1.30 1.44 1.43 1.28 0.71 304 A304P 1.24 1.30 1.24 1.35 1.49 1.34 0.77 304 A304R 1.14 1.16 1.05 1.13 0.93 1.03 0.90 304 A304S 0.84 0.94 0.80 1.15 0.87 0.71 0.67 304 A304T 1.00 1.07 0.89 0.99 0.98 0.75 0.92 304 A304V 1.16 1.18 1.13 1.21 0.94 0.70 0.81 304 A304W 0.88 0.89 0.89 0.93 0.73 0.94 1.07 304 A304Y 0.89 0.93 0.87 0.95 0.66 0.77 1.01 308 R308A 1.52 1.58 1.23 1.51 1.40 1.34 0.52 308 R308C 1.61 2.01 0.97 1.22 1.95 1.87 0.39 308 R308D 2.54 2.88 1.75 2.01 3.24 2.75 0.24 308 R308E 1.54 1.66 1.15 1.34 1.42 1.45 0.53 308 R308F 1.91 2.67 0.69 1.30 1.80 1.55 0.18 308 R308G 1.27 1.29 0.81 0.95 1.02 1.10 0.59 308 R308H 1.10 1.08 0.77 0.92 1.06 0.97 0.66 308 R308I 1.13 1.55 0.57 0.76 0.92 0.84 0.43 308 R308K 1.00 0.97 1.00 0.99 0.99 0.92 0.95 308 R308L 0.97 1.33 0.36 0.67 0.93 0.19 0.33 308 R308M 1.94 2.07 1.31 1.66 1.65 1.92 0.38 308 R308N 1.72 1.86 1.09 1.37 1.94 1.71 0.41 308 R308P 4.38 4.55 1.93 2.52 5.05 3.89 0.12 308 R308S 1.06 1.00 0.84 0.97 0.84 0.87 1.00 308 R308T 1.36 1.34 0.91 1.08 1.16 1.24 0.73 308 R308V 1.31 1.47 0.62 0.88 1.06 0.97 0.42 308 R308W 0.92 1.50 0.34 0.70 1.29 1.01 0.31 308 R308Y 0.90 1.28 0.46 0.70 0.92 1.14 0.41 321 T321A 1.07 1.20 0.95 1.19 1.44 1.33 0.84 321 T321C 1.35 1.36 0.92 1.09 1.63 1.10 0.52 321 T321F 1.00 0.96 0.90 1.00 1.14 1.07 1.03 321 T321H 0.89 0.85 0.81 0.90 1.03 1.37 1.14 321 T321I 0.81 0.87 0.66 0.77 1.13 0.93 0.83 321 T321L 0.74 0.77 0.67 0.75 0.91 0.96 1.11 321 T321P 1.23 1.25 1.08 1.18 1.69 1.22 0.79 321 T321Q 1.12 1.15 1.05 1.10 1.14 1.27 0.93 321 T321R 1.02 0.92 1.02 0.96 1.09 0.96 1.04 321 T321S 1.02 0.91 0.88 0.99 1.14 0.84 1.08 321 T321V 0.95 0.92 0.72 0.81 1.34 0.43 0.84 321 T321Y 0.76 0.82 0.69 0.77 1.23 0.79 1.04 358 Q358A 1.07 1.42 1.63 1.45 1.54 1.31 0.65 358 Q358C 1.95 2.35 1.34 1.85 2.83 1.62 0.26 358 Q358D 1.35 1.37 1.21 1.29 1.55 1.10 0.79 358 Q358E 1.30 1.35 1.22 1.27 1.57 1.02 0.74 358 Q358F 1.35 1.32 1.05 1.14 1.49 1.19 0.71 358 Q358G 1.15 1.03 0.90 0.96 1.10 1.06 0.74 358 Q358H 1.05 0.99 0.95 0.97 1.15 1.40 0.94 358 Q358L 0.92 1.06 1.05 1.06 1.24 0.84 0.83 358 Q358M 1.12 1.37 1.42 1.41 1.52 1.17 0.72 358 Q358N 1.27 1.37 1.32 1.42 1.63 1.06 0.73 358 Q358P 1.27 1.33 1.10 1.23 1.72 1.17 0.65 358 Q358R 1.03 1.07 1.08 1.07 1.18 1.15 0.91 358 Q358S 1.09 0.99 1.02 1.04 1.01 0.91 0.93 358 Q358T 1.05 1.05 1.04 1.00 1.17 1.10 0.95 358 Q358V 1.15 1.18 1.02 1.13 1.35 1.35 0.67 378 P378C 28.49 39.29 11.48 19.82 40.84 34.77 0.05 378 P378D 1.13 1.20 1.15 1.18 1.12 0.99 0.88 378 P378F 1.84 2.17 0.62 1.01 2.97 0.36 0.15 378 P378G 1.21 1.20 1.00 1.06 1.44 1.30 0.75 378 P378H 0.90 0.94 0.80 0.85 1.11 0.50 1.06 378 P378I 1.16 1.20 1.03 1.15 1.33 1.26 0.62 378 P378L 0.78 0.89 0.88 0.97 1.08 0.79 0.82 378 P378N 1.31 1.39 1.19 1.37 1.42 0.97 0.71 378 P378R 0.94 0.93 1.10 1.07 1.05 1.03 1.28 378 P378S 1.15 1.09 0.99 1.04 1.08 1.11 0.90 378 P378T 0.83 0.96 0.90 0.93 0.87 0.73 1.04 378 P378V 1.08 1.08 1.09 1.10 1.15 0.94 0.87 378 P378Y 0.87 0.92 0.78 0.91 1.11 0.97 0.67 382 S382A 1.05 1.33 1.16 1.30 1.57 1.17 0.76 382 S382C 1.20 1.19 1.01 1.12 1.27 1.05 0.87 382 S382D 1.43 1.42 1.19 1.34 1.35 1.41 0.74 382 S382E 1.23 1.39 1.14 1.27 1.47 1.23 0.83 382 S382G 1.05 1.06 0.90 0.96 0.96 0.95 1.02 382 S382H 1.10 1.02 0.96 0.99 1.10 0.98 1.00 382 S382I 1.03 1.07 0.82 0.88 1.07 0.96 0.79 382 S382K 0.94 0.84 0.95 0.98 1.12 0.78 1.09 382 S382L 0.93 0.93 0.69 0.82 0.95 0.70 0.79 382 S382M 1.51 1.84 1.25 1.51 2.20 1.47 0.54 382 S382N 1.39 1.43 1.15 1.35 1.55 1.16 0.72 382 S382P 1.41 1.42 1.22 1.33 1.65 1.25 0.70 382 S382R 1.23 1.19 0.97 1.17 1.14 0.78 0.91 382 S382T 1.18 1.15 0.98 1.08 1.30 1.14 0.90 382 S382V 1.13 1.07 0.85 0.98 1.22 0.89 0.81 382 S382W 1.08 1.03 0.90 0.98 1.19 0.94 0.98 383 K383A 1.26 1.16 1.24 1.35 1.17 0.86 0.66 383 K383C 1.85 2.15 0.60 1.01 1.16 0.02 0.16 383 K383D 1.14 1.17 1.16 1.13 1.04 0.89 0.69 383 K383E 1.04 0.98 0.98 1.00 1.00 0.82 0.94 383 K383F 1.43 1.44 0.93 1.01 1.47 0.86 0.50 383 K383H 0.80 0.78 0.79 0.84 0.97 1.03 0.96 383 K383L 0.64 0.66 0.50 0.60 0.66 0.92 0.76 383 K383M 1.32 1.19 0.94 1.10 1.20 1.01 0.57 383 K383N 1.27 1.20 0.98 1.11 1.07 1.16 0.64 383 K383P 1.89 2.46 1.17 1.37 1.82 2.31 0.22 383 K383Q 1.03 0.98 1.01 1.06 0.95 1.15 0.94 383 K383R 1.02 0.91 1.00 0.95 1.10 0.84 0.99 383 K383S 0.92 0.92 0.87 0.92 0.97 0.74 0.86 383 K383T 0.79 0.76 0.83 0.84 0.90 0.83 0.98 383 K383W 0.70 0.78 0.62 0.68 0.74 0.39 0.61 383 K383Y 0.62 0.72 0.78 0.75 0.73 0.67 0.87 398 T398A 1.28 1.18 1.34 1.43 1.37 1.17 0.73 398 T398C 1.58 1.46 1.20 1.39 1.61 1.35 0.46 398 T398D 1.29 1.24 1.33 1.33 0.88 1.42 0.75 398 T398E 1.33 1.24 1.31 1.32 1.17 1.38 0.81 398 T398I 0.81 0.87 0.91 0.96 0.91 0.86 1.04 398 T398K 0.92 0.76 0.99 0.97 0.97 0.66 1.06 398 T398L 0.76 0.68 0.85 0.87 0.86 1.14 0.99 398 T398M 1.29 1.24 1.39 1.38 1.18 1.03 0.75 398 T398N 1.96 1.88 1.34 1.58 1.23 1.31 0.37 398 T398P 1.38 1.29 1.33 1.35 0.97 1.29 0.77 398 T398Q 1.47 1.41 1.61 1.52 1.46 1.51 0.74 398 T398R 1.16 1.08 1.25 1.16 1.25 1.02 0.98 398 T398S 1.03 0.94 1.08 1.05 0.92 0.67 1.07 398 T398V 1.00 1.06 1.20 1.19 1.03 0.96 0.87 405 H405A 1.61 1.64 1.45 1.54 1.54 1.15 0.56 405 H405C 1.92 2.06 1.69 1.91 2.68 1.40 0.33 405 H405D 1.45 1.44 1.25 1.30 1.37 1.21 0.47 405 H405F 2.03 2.09 1.28 1.41 3.15 1.98 0.20 405 H405G 1.16 1.20 1.10 1.14 1.50 1.14 0.72 405 H405K 0.71 0.58 0.87 0.89 1.05 0.64 0.72 405 H405L 0.79 0.89 0.77 0.94 0.97 0.72 0.49 405 H405M 1.65 1.66 1.28 1.51 1.67 1.22 0.41 405 H405N 1.42 1.26 1.37 1.42 1.43 1.09 0.69 405 H405P −12.67 −11.60 −0.88 −2.21 −6.12 −0.26 −0.03 405 H405Q 1.50 1.53 1.33 1.42 1.76 1.33 0.59 405 H405R 1.54 1.56 1.22 1.36 1.29 0.84 0.51 405 H405S 1.09 1.00 1.05 1.03 1.10 0.91 0.90 405 H405T 0.99 0.94 0.86 0.89 0.96 0.77 0.87 405 H405W 0.86 0.83 0.43 0.64 1.32 0.50 0.35 405 H405Y 0.96 1.07 0.84 0.93 1.37 0.96 0.50 417 T417A 1.55 1.30 1.42 1.39 1.28 1.05 0.71 417 T417D 1.55 1.28 1.40 1.43 1.14 1.34 0.80 417 T417E 1.22 1.07 1.16 1.17 1.12 1.07 0.89 417 T417H 0.98 0.89 0.91 0.95 1.07 0.89 0.98 417 T417I 1.00 0.99 0.79 0.84 0.90 0.82 0.82 417 T417L 0.87 0.79 0.88 0.89 0.98 1.03 0.92 417 T417M 1.65 1.65 1.39 1.52 1.82 1.41 0.49 417 T417P 1.38 1.25 1.34 1.35 1.26 1.12 0.74 417 T417Q 1.47 1.24 1.21 1.24 1.40 1.26 0.84 417 T417R 1.34 1.15 1.36 1.31 1.13 1.08 0.85 417 T417S 1.10 0.95 1.01 1.03 0.96 1.13 1.02 417 T417V 0.88 0.86 0.85 0.92 0.97 0.94 0.97 417 T417W 0.97 0.84 0.72 0.89 1.01 0.77 0.90 418 E418A 1.16 1.16 1.32 1.29 1.44 0.99 0.83 418 E418C 1.14 1.24 0.98 1.10 1.21 0.73 0.66 418 E418D 0.94 0.94 1.06 1.11 0.98 0.84 0.96 418 E418G 0.68 0.70 0.81 0.82 0.82 0.58 1.17 418 E418H 0.71 0.73 0.76 0.80 0.87 0.75 1.04 418 E418I 0.80 0.75 0.76 0.79 1.09 0.70 0.97 418 E418K 0.68 0.62 0.68 0.71 0.77 0.79 1.27 418 E418L 0.69 0.64 0.72 0.78 0.39 0.47 1.08 418 E418M 0.96 0.95 1.01 1.07 1.33 0.81 0.86 418 E418N 1.00 1.01 1.18 1.20 1.11 0.87 0.83 418 E418P 1.04 1.03 1.15 1.19 1.09 0.72 0.87 418 E418Q 1.11 1.13 1.05 1.16 1.22 0.76 0.84 418 E418R 1.08 1.01 0.95 1.03 1.26 1.01 0.86 418 E418S 1.03 0.94 0.80 0.85 0.94 0.85 0.86 418 E418T 0.87 0.80 0.82 0.84 0.91 0.50 1.09 418 E418V 0.71 0.65 0.66 0.82 0.83 0.34 1.22 418 E418Y 0.80 0.77 0.65 0.79 0.88 0.59 0.81 420 P420A 1.16 1.17 1.41 1.49 1.36 0.97 0.81 420 P420C 1.17 1.32 1.37 1.47 1.09 1.02 0.70 420 P420D 1.29 1.25 1.37 1.38 1.35 0.90 0.75 420 P420E 1.32 1.27 1.32 1.35 1.45 0.79 0.75 420 P420H 1.05 0.95 0.95 1.01 1.11 0.69 0.98 420 P420I 0.98 0.95 0.87 0.95 1.07 0.62 0.98 420 P420L 0.85 0.82 0.87 0.91 1.07 0.74 0.97 420 P420M 1.37 1.41 1.33 1.29 1.28 0.75 0.73 420 P420N 1.42 1.38 1.30 1.41 1.50 0.95 0.73 420 P420R 0.49 −5.15 −1.38 −0.61 −11.09 −1.99 −0.01 420 P420S 1.01 0.88 1.10 1.09 0.93 0.81 1.11 420 P420T 0.97 0.87 0.94 0.95 0.68 0.87 1.10 420 P420V 1.11 1.06 1.10 1.12 1.21 0.77 0.84 420 P420W 0.72 0.69 0.71 0.75 0.80 0.54 1.29 420 P420Y 0.90 0.83 0.80 0.76 1.05 0.85 0.96 421 G421A 1.36 1.28 1.23 1.30 1.31 0.79 0.79 421 G421D 1.23 1.28 1.43 1.38 1.28 0.99 0.72 421 G421E 1.44 1.43 1.47 1.46 1.48 1.23 0.66 421 G421F 1.23 1.19 1.13 1.13 1.50 0.75 0.79 421 G421H 0.91 0.81 0.80 0.84 0.69 0.71 1.19 421 G421I 1.19 1.13 0.97 1.08 1.38 0.80 0.65 421 G421L 0.80 0.77 0.91 0.95 1.24 0.74 0.97 421 G421N 1.28 1.30 1.23 1.34 1.49 0.82 0.75 421 G421P 1.20 1.22 1.22 1.27 1.41 0.83 0.74 421 G421Q 1.31 1.27 1.27 1.29 1.30 1.15 0.75 421 G421R 1.17 1.04 1.05 1.09 1.08 0.85 1.00 421 G421S 1.11 1.02 1.05 1.04 1.05 0.80 0.97 421 G421T 1.04 0.91 0.95 0.98 1.05 0.86 1.05 421 G421W 0.84 0.85 0.86 0.94 0.94 0.74 0.96 421 G421Y 0.98 0.86 0.89 0.94 1.12 0.64 0.95 432 P432A 1.40 1.30 1.44 1.53 1.64 1.26 0.75 432 P432D 1.77 1.59 1.72 1.77 1.95 1.24 0.61 432 P432E 1.39 1.29 1.40 1.41 1.33 1.19 0.77 432 P432H 1.14 1.07 0.83 1.01 1.35 0.97 0.80 432 P432K 1.21 0.96 1.05 1.08 1.22 1.02 0.91 432 P432L 1.06 0.90 0.98 0.98 1.26 0.63 0.97 432 P432M 1.48 1.47 1.72 1.71 2.15 1.45 0.65 432 P432N 1.31 1.38 1.28 1.41 1.59 1.02 0.73 432 P432Q 1.62 1.39 1.31 1.46 1.69 1.46 0.70 432 P432R 1.60 1.35 1.36 1.43 1.27 1.21 0.78 432 P432S 1.16 1.02 0.94 1.00 1.07 0.78 1.04 432 P432T 1.19 0.99 1.33 1.13 1.27 1.08 0.98 432 P432Y 1.14 0.98 0.82 0.94 1.21 0.69 0.90 437 W437C 0.83 0.77 1.09 1.15 0.89 0.84 1.01 437 W437D 0.97 0.85 1.12 1.15 0.98 0.97 1.08 437 W437E 0.99 1.00 0.79 0.87 0.95 0.76 0.59 437 W437F 1.01 0.81 0.94 1.00 0.74 0.80 1.22 437 W437G 0.83 0.72 0.82 0.82 0.78 0.66 1.42 437 W437H 0.65 0.64 0.83 0.84 0.78 0.71 1.52 437 W437L 0.64 0.60 0.73 0.75 0.59 0.68 1.55 437 W437M 1.03 0.86 1.01 1.06 0.83 0.63 1.11 437 W437N 1.01 0.94 1.02 1.06 0.87 1.07 1.04 437 W437Q 1.05 0.90 1.08 1.11 0.90 0.77 1.12 437 W437R 0.91 0.83 1.07 1.02 0.83 0.53 1.19 437 W437S 0.96 0.75 0.99 0.96 0.86 0.90 1.24 437 W437T 0.78 0.71 0.92 0.89 0.77 0.71 1.32 437 W437V 0.75 0.73 0.90 0.87 0.81 0.87 1.36 437 W437Y 0.66 0.59 0.78 0.77 0.64 0.68 1.50 443 Q443A 1.24 1.01 1.35 1.32 1.08 1.08 0.83 443 Q443C 1.27 1.23 1.16 1.22 1.02 1.22 0.80 443 Q443F 1.26 1.14 1.18 1.20 1.10 1.15 0.86 443 Q443G 1.12 0.94 1.11 1.02 1.01 1.00 1.08 443 Q443K 0.79 0.75 0.93 0.90 0.83 0.76 1.10 443 Q443L 0.84 0.81 1.07 1.02 0.89 0.85 1.09 443 Q443N 1.22 1.18 1.41 1.53 1.21 1.23 0.77 443 Q443P 1.02 0.97 1.08 1.16 1.03 0.64 0.92 443 Q443R 1.06 1.03 1.03 1.10 0.99 0.98 0.98 443 Q443S 0.89 0.82 0.95 0.94 0.84 0.87 1.20 443 Q443T 1.01 0.76 0.88 0.89 0.89 0.69 1.15 443 Q443V 1.02 0.90 1.09 1.10 1.23 1.03 0.95 443 Q443W 0.57 0.59 0.57 0.63 0.65 0.63 0.87 443 Q443Y 0.82 0.87 0.95 0.97 0.97 0.86 0.92 446 G446A 1.00 0.91 1.25 1.26 0.97 1.08 0.95 446 G446C 1.39 1.31 1.84 1.78 1.40 1.23 0.73 446 G446D 1.12 0.99 1.15 1.25 1.15 0.97 0.93 446 G446F 1.34 1.37 1.00 1.15 1.03 1.22 0.75 446 G446H 0.93 0.80 0.65 0.70 0.84 0.83 1.32 446 G446I 1.22 1.07 0.97 1.04 1.39 1.05 0.70 446 G446K 0.97 0.83 0.80 0.86 0.70 0.87 1.10 446 G446L 0.80 0.78 0.86 0.88 0.94 0.85 1.07 446 G446M 1.21 1.05 0.85 0.99 1.06 1.16 0.87 446 G446N 1.17 1.08 1.05 1.21 1.07 1.07 0.88 446 G446P 1.21 1.09 1.07 1.16 1.09 0.95 0.74 446 G446Q 1.28 1.21 1.16 1.23 1.09 1.01 0.83 446 G446R 0.37 0.07 0.71 1.10 0.22 0.65 0.75 446 G446S 1.07 0.95 0.95 0.95 0.75 1.01 1.11 446 G446T 1.03 0.88 0.69 0.81 0.91 0.96 1.09 446 G446V 1.17 1.09 0.91 0.98 1.14 0.90 0.82 446 G446W 0.85 0.72 0.83 0.89 0.92 0.80 1.05 446 G446Y 0.88 0.76 1.02 1.00 0.83 0.91 1.09 454 G454A 1.33 1.25 1.61 1.60 1.14 1.26 0.88 454 G454C 1.33 1.18 1.15 1.20 1.14 1.37 0.78 454 G454D 1.40 1.30 1.27 1.36 1.18 1.09 0.76 454 G454E 1.36 1.21 1.08 1.19 0.99 0.91 0.83 454 G454H 0.92 0.83 0.89 0.95 0.83 0.83 1.10 454 G454I 0.87 0.79 0.77 0.82 0.88 0.75 1.02 454 G454K 0.86 0.80 0.99 0.97 0.82 0.81 1.13 454 G454L 0.12 −0.34 −0.62 −0.26 0.50 −0.62 0.11 454 G454M 1.39 1.26 1.14 1.42 1.26 1.30 0.68 454 G454N 1.20 1.09 1.07 1.20 1.11 1.10 0.91 454 G454P 1.41 1.34 1.14 1.29 1.16 0.96 0.77 454 G454R 1.25 1.09 0.99 1.12 1.07 1.01 0.92 454 G454S 0.83 0.80 0.83 0.90 0.89 0.79 1.17 454 G454T 1.04 0.93 0.90 0.98 0.99 0.94 1.02 454 G454V 1.20 1.07 1.05 1.10 1.18 0.89 0.93 457 S457A 1.05 0.95 1.22 1.27 1.23 1.21 0.89 457 S457C 1.27 1.23 0.72 0.72 1.53 1.44 0.59 457 S457D 1.02 0.89 1.05 1.16 0.93 0.81 0.99 457 S457E 1.10 0.95 0.97 1.06 0.97 0.79 0.95 457 S457G 0.82 0.71 0.82 0.87 0.80 0.88 1.21 457 S457H 0.83 0.72 0.81 0.90 0.89 1.12 1.17 457 S457K 0.74 0.63 0.79 0.86 0.76 0.62 1.37 457 S457L 0.67 0.61 0.68 0.79 0.64 0.63 1.27 457 S457M 1.07 0.98 0.96 1.08 1.03 0.99 0.92 457 S457N 1.08 0.92 1.10 1.19 1.13 1.15 0.86 457 S457P 1.29 1.21 1.09 1.18 1.30 1.19 0.88 457 S457Q 1.10 1.01 1.06 1.13 1.14 0.96 0.91 457 S457R 1.58 1.31 0.89 1.10 1.59 1.89 0.42 457 S457T 0.88 0.70 0.94 0.94 0.85 1.01 1.18 457 S457V 0.89 0.82 0.83 0.88 0.84 0.78 1.11 457 S457W 0.87 0.69 0.64 0.74 0.84 0.60 1.05 457 S457Y 0.81 0.70 0.70 0.80 0.80 0.79 1.15 459 T459A 1.10 1.03 1.25 1.34 1.46 1.18 0.83 459 T459D 1.20 1.17 1.19 1.24 1.37 1.44 0.82 459 T459G 1.15 1.01 0.98 0.97 0.81 1.36 1.08 459 T459I 1.05 0.95 0.95 1.01 1.04 1.04 1.02 459 T459K 1.04 0.91 0.86 0.90 0.80 0.72 1.08 459 T459L 0.93 0.83 1.19 1.10 0.91 0.81 1.04 459 T459Q 1.46 1.43 1.40 1.47 1.29 1.24 0.72 459 T459R 1.09 1.00 1.09 1.09 0.90 0.97 1.05 459 T459S 0.99 1.00 0.85 0.87 1.04 0.90 1.06 459 T459V 1.11 1.02 0.92 0.99 1.07 1.35 0.93 459 T459Y 1.10 1.02 0.97 1.06 1.14 0.74 0.92 461 T461A 1.40 1.44 1.16 1.28 1.74 1.18 0.47 461 T461D 1.26 1.09 1.21 1.25 1.25 1.10 0.92 461 T461E 1.52 1.44 1.49 1.43 1.22 1.06 0.74 461 T461F 1.29 1.22 1.31 1.25 1.14 1.12 0.79 461 T461G 1.09 1.03 1.08 1.04 1.16 1.14 0.98 461 T461I 1.11 1.00 0.99 1.05 1.03 1.33 0.93 461 T461K 0.89 0.75 0.78 0.91 0.98 0.99 1.01 461 T461L 0.92 0.85 1.03 1.02 1.15 0.91 0.87 461 T461N 1.18 1.18 1.35 1.37 1.36 1.30 0.81 461 T461P 1.19 1.10 1.37 1.33 1.14 1.17 0.92 461 T461R 1.19 1.04 1.13 1.18 1.11 0.91 0.99 461 T461S 1.12 1.04 0.87 0.88 1.03 0.90 0.88 461 T461V 0.95 1.00 1.01 1.05 1.11 1.28 0.87 461 T461W 0.97 0.82 0.77 0.86 0.75 0.67 0.87 461 T461Y 1.02 0.93 0.90 1.02 1.01 1.11 0.87 464 S464D 1.45 1.23 1.24 1.31 1.51 1.21 0.73 464 S464E 0.95 0.98 1.04 1.06 1.06 1.23 1.03 464 S464G 0.94 0.86 0.92 1.00 0.98 0.97 1.04 464 S464H 0.88 0.84 0.82 0.87 0.99 1.04 1.10 464 S464I 0.94 0.80 0.78 0.83 0.96 1.15 1.00 464 S464K 0.94 0.85 0.88 0.93 0.93 0.81 0.97 464 S464L 0.77 0.81 0.79 0.91 0.88 0.86 1.02 464 S464M 1.32 1.27 1.35 1.50 1.18 1.42 0.81 464 S464N 1.15 1.03 1.16 1.25 1.18 1.38 0.85 464 S464P 1.42 1.38 1.32 1.42 1.40 1.56 0.76 464 S464Q 1.33 1.30 1.12 1.29 1.28 1.21 0.80 464 S464V 0.94 0.96 0.94 1.02 1.12 1.33 0.89 464 S464W 1.06 0.95 0.94 1.08 0.98 0.84 1.01 464 S464Y 0.81 0.71 1.01 1.02 0.75 0.84 1.33 474 G474A 1.03 1.20 1.25 1.35 1.12 1.27 0.81 474 G474C 1.05 1.30 1.18 1.31 1.55 1.04 0.66 474 G474D 1.13 1.26 1.35 1.41 1.26 1.28 0.78 474 G474E 1.13 1.13 1.17 1.27 1.16 1.23 0.87 474 G474F 1.23 1.34 1.20 1.29 1.22 1.47 0.80 474 G474H 0.86 0.95 0.94 1.02 1.06 1.11 1.07 474 G474I 0.79 0.99 0.91 0.97 0.95 0.81 0.99 474 G474K 0.81 0.82 0.92 0.97 1.06 0.97 1.07 474 G474L 0.70 0.75 0.81 0.87 0.88 0.91 1.12 474 G474M 1.18 1.26 1.24 1.40 1.20 1.05 0.74 474 G474N 1.14 1.02 1.09 1.16 1.29 1.29 0.89 474 G474P 1.53 1.45 1.25 1.38 1.39 0.36 0.72 474 G474Q 1.19 1.14 1.26 1.33 1.35 1.37 0.79 474 G474R 1.15 1.17 1.16 1.29 1.31 1.41 0.82 474 G474S 0.87 0.89 0.97 0.99 1.00 0.93 1.09 474 G474T 0.97 1.01 1.13 1.06 1.03 1.00 1.01 474 G474V 0.88 0.91 1.03 1.02 1.03 0.94 1.01 483 R483A 1.25 1.23 1.35 1.43 1.43 1.34 0.74 483 R483C 1.19 1.17 1.73 1.51 1.19 1.59 0.70 483 R483F 1.08 1.49 1.36 1.23 1.51 1.18 0.57 483 R483G 1.07 1.13 1.07 1.12 1.14 1.08 0.86 483 R483K 0.82 0.73 0.86 0.90 0.91 0.69 1.12 483 R483L 1.10 1.03 1.29 1.20 1.06 0.93 0.94 483 R483M 1.31 1.50 1.42 1.56 1.51 1.26 0.66 483 R483N 1.17 1.32 1.46 1.39 1.34 1.09 0.75 483 R483P 1.19 1.17 1.37 1.35 1.17 1.05 0.83 483 R483Q 1.38 1.48 1.84 1.67 1.72 1.51 0.65 483 R483S 0.99 1.05 0.92 0.96 1.04 0.94 1.02 483 R483T 1.10 1.03 1.04 0.98 0.98 0.93 0.93 483 R483V 1.11 1.18 1.25 1.26 1.25 1.20 0.80 483 R483Y 0.81 0.86 1.07 1.06 1.04 0.81 0.96

It will be apparent that a large number of substitutions produced variant α-amylases having one or more improved properties compared to the wild-type α-amylase. These substitutions are included in the present compositions and methods.

Example 24 Altered Property of AmyS Variants

This example shows that some G. stearothermophilus α-amylase (AmyS) variants (described in Example 22) have an altered property relative to the parent α-amylase. A high throughput thermal stability screen of AmyS variants was carried out as described in Example 3. Performance indices for activity (measured as BODIPY assay) and residual activity (after thermal stress) are shown in Tables 24-1, 24-2, 24-3.

TABLE 24-1 Positions in AmyS protein with mutations (shown in column labeled variant) that have performance indices for both activity and residual activity after heat stress better than wildtype AmyS. Residual Variant Activity Activity  72 D 1.0300361 1.0701945  74 A 1.0821966 1.2443197  74 E 1.0425346 1.0348158  74 G 1.1272413 1.0873821  74 H 1.0489031 1.0047597  74 I 1.0441329 1.0204416  74 Y 1.0962024 1.0243028  86 E 1.0744425 1.0082956  86 K 1.0320588 1.0003874 115 E 1.0371929 1.0263233 115 K 1.0983963 1.1179136 115 L 1.0304852 1.2220073 115 N 1.0617811 1.0373701 115 Q 1.0585819 1.0086197 115 R 1.0362569 1.0196812 115 Y 1.0818022 1.0155115 124 A 1.0066154 1.0764058 124 K 1.0875013 1.3977188 124 N 1.2073767 1.1957849 124 Q 1.0581528 1.1748222 124 R 1.0401245 1.2046408 125 N 1.2111343 1.111989 132 A 1.0229275 1.3339209 135 F 1.0125922 1.1400675 145 A 1.0535347 1.269397 146 A 1.0159296 1.2695343 146 D 1.0111003 1.0989512 146 E 1.0209598 1.076157 146 T 1.0557131 1.0905141 146 W 1.0503671 1.0446909 148 A 1.1550962 1.3714229 148 E 1.0257123 1.1367377 148 F 1.0047267 1.0833811 148 R 1.1439066 1.0724962 153 A 1.0933406 1.1903028 153 D 1.047218 1.0763254 153 G 1.0314126 1.0393344 153 H 1.0243746 1.0325474 153 N 1.0749307 1.1537286 153 P 1.1090313 1.1653113 153 R 1.0695773 1.050884 159 A 1.2514424 1.8489959 159 C 1.1389324 1.4229765 159 D 1.3042895 1.616517 159 E 1.3048703 1.6426287 159 F 1.0692526 1.2740874 159 G 1.309088 1.4806394 159 H 1.2239861 1.4766606 159 K 1.3024788 1.6188749 159 L 1.2438467 1.7685564 159 N 1.4021695 1.747298 159 R 1.3445318 1.6062932 159 S 1.3352659 1.5322275 159 T 1.2115923 1.5982316 159 V 1.1075763 1.5364844 169 L 1.2709 1.221157 169 M 1.0720854 1.2525822 169 Y 1.1519097 1.3009779 179 A 1.2856782 1.4150905 179 Q 1.0837406 1.0777175 180 A 1.223674 1.1463487 181 A 1.5853606 2.5498838 181 C 1.0805237 1.2359592 181 D 1.2451756 1.4958763 181 E 1.2126846 1.3673333 181 F 1.1174172 1.0714025 181 L 1.0562715 1.2603028 181 M 1.0553459 1.1115696 181 N 1.0657087 1.058626 181 P 1.3407541 1.8191875 181 Q 1.1827757 1.3094913 181 R 2.1023852 1.000651 181 V 1.2072805 1.2882775 181 Y 1.1468422 1.2888335 187 L 1.0631177 1.1713174 242 D 1.053295 1.2659451 242 E 1.1904636 1.4089496 242 G 1.0897161 1.0670134 259 M 1.054788 1.1174398 261 L 1.1311136 1.2682418 271 K 1.0660617 1.1281026 271 V 1.0912656 1.3024768 278 A 1.1681249 1.3749858 278 H 1.2287582 1.3214257 278 K 1.2908668 1.3351968 278 N 1.2587781 1.4816971 278 R 1.2602246 1.3802029 278 S 1.0407916 1.051006 281 A 1.0778757 1.302493 281 I 1.0773434 1.0691046 281 L 1.0664433 1.5428781 281 M 1.2357293 1.317267 281 P 1.1452343 1.1634661 281 R 1.1498741 1.1898966 281 Y 1.0366211 1.0814182 302 C 1.0627926 1.0743991 302 D 1.3067743 1.3085968 302 E 1.0492343 1.1151221 302 M 1.0807557 1.2463993 304 D 1.1358974 1.304862 304 E 1.1872403 1.2138013 304 F 1.1250781 1.0476505 304 M 1.2316987 1.2224245 304 N 1.0270711 1.0584592 304 P 1.0166456 1.0403283 304 R 1.0960387 1.0336549 304 V 1.0716606 1.0416779 304 W 1.1600113 1.0109269 304 Y 1.3289811 1.1964204 308 A 1.0074309 1.189004 321 A 1.0826055 1.2311805 321 H 1.384587 1.4691649 321 Q 1.3306703 1.3485614 321 R 1.2446359 1.3138378 321 S 1.1483705 1.1251132 321 Y 1.0396471 1.1263643 333 Q 1.425789 1.6656427 378 D 1.0880667 1.2202146 378 N 1.0064817 1.2616767 378 R 1.0264777 1.2826859 378 T 1.042994 1.0795534 382 D 1.1628676 1.2206133 382 G 1.0050534 1.009576 382 K 1.1896345 1.178075 382 N 1.0241429 1.1576205 382 P 1.001145 1.0672392 398 A 1.0127464 1.2067063 418 A 1.070915 1.3701437 418 M 1.101424 1.3091549 418 N 1.1440828 1.4650527 420 A 1.1288416 1.2216203 420 D 1.0368387 1.065286 420 M 1.011372 1.1274183 420 N 1.0213745 1.1440374 421 E 1.010536 1.0961403 421 H 1.0434891 1.0576175 421 L 1.0197128 1.0679988 421 N 1.092512 1.1299631 421 Q 1.0784982 1.1126707 421 R 1.142674 1.2396538 421 T 1.0098565 1.023113 432 A 1.1828859 1.4534375 432 D 1.1261465 1.2701694 432 E 1.0932052 1.1438228 432 K 1.0432215 1.1145887 432 L 1.1040571 1.2896033 432 M 1.1530369 1.3947422 432 N 1.1373288 1.2843802 432 Q 1.2305257 1.3438957 432 R 1.1226193 1.2108348 432 S 1.1383528 1.1690319 432 T 1.0946975 1.1651163 432 Y 1.0242088 1.2209025 437 C 1.0389223 1.0550093 437 D 1.0648095 1.2263069 437 F 1.0884138 1.0761389 437 G 1.1270339 1.2057266 437 H 1.0624587 1.2128077 437 L 1.0706178 1.2702869 437 M 1.1727007 1.3357945 437 N 1.0678835 1.1245993 437 Q 1.0533035 1.1845926 437 R 1.0211609 1.01587 437 S 1.0996009 1.0837657 437 V 1.0035949 1.1373234 437 Y 1.2190374 1.428939 443 G 1.039287 1.0340347 443 L 1.0229234 1.0966951 443 P 1.0219417 1.0948128 446 A 1.2002798 1.498028 446 D 1.0773299 1.1176728 446 H 1.0897531 1.071114 446 K 1.039616 1.0263734 446 N 1.1867752 1.1501356 446 R 1.0179243 1.035122 446 S 1.00426 1.0219768 446 Y 1.1205486 1.2525673 459 I 1.0404304 1.0379194 459 M 1.0320006 1.1066777 459 Y 1.0131462 1.0198803 461 P 1.084833 1.1869717 464 D 1.0164453 1.0297077 464 H 1.0355113 1.0962268 464 L 1.0084324 1.0510274 464 M 1.0026999 1.1589373 464 N 1.0727228 1.1205096 464 Q 1.0719588 1.2199585 464 Y 1.1888873 1.3747167 474 A 1.1556971 1.3935021 474 D 1.0692943 1.1879003 474 E 1.1729152 1.3481142 474 F 1.0633952 1.1462803 474 H 1.0620029 1.1722857 474 I 1.0766474 1.1352128 474 K 1.1240341 1.2036886 474 L 1.110407 1.267509 474 M 1.1869843 1.3422689 474 N 1.1135684 1.2124349 474 P 1.0761861 1.2293237 474 Q 1.2580448 1.3477339 474 R 1.1994238 1.3506214 474 S 1.2348915 1.2615358 474 T 1.1757697 1.1841873 474 V 1.0823992 1.2078523

TABLE 24-2 Positions in AmyS protein with mutations (shown in column labeled variant) that have performance indices for residual activity after heat stress at least 20% better than wildtype AmyS and performance indices for starting activity or expression at least half of wildtype AmyS. Residual Variant Activity Activity  74A 1.0821966 1.2443197 115L 1.0304852 1.2220073 124K 1.0875013 1.3977188 124R 1.0401245 1.2046408 132A 1.0229275 1.3339209 132C 0.9072598 1.2271522 135A 0.9014583 1.2604591 145A 1.0535347 1.269397 146A 1.0159296 1.2695343 148A 1.1550962 1.3714229 148N 0.8803735 1.202166 159A 1.2514424 1.8489959 159C 1.1389324 1.4229765 159D 1.3042895 1.616517 159E 1.3048703 1.6426287 159F 1.0692526 1.2740874 159G 1.309088 1.4806394 159H 1.2239861 1.4766606 159K 1.3024788 1.6188749 159L 1.2438467 1.7685564 159N 1.4021695 1.747298 159R 1.3445318 1.6062932 159S 1.3352659 1.5322275 159T 1.2115923 1.5982316 159V 1.1075763 1.5364844 169A 0.9976004 1.3149706 169L 1.2709 1.221157 169M 1.0720854 1.2525822 169Y 1.1519097 1.3009779 179A 1.2856782 1.4150905 181A 1.5853606 2.5498838 181C 1.0805237 1.2359592 181D 1.2451756 1.4958763 181E 1.2126846 1.3673333 181L 1.0562715 1.2603028 181P 1.3407541 1.8191875 181Q 1.1827757 1.3094913 181V 1.2072805 1.2882775 181Y 1.1468422 1.2888335 242A 0.8658592 1.3402797 242D 1.053295 1.2659451 242E 1.1904636 1.4089496 242Q 0.9905304 1.8848517 261L 1.1311136 1.2682418 271A 0.9883235 1.3367718 271V 1.0912656 1.3024768 278A 1.1681249 1.3749858 278H 1.2287582 1.3214257 278K 1.2908668 1.3351968 278N 1.2587781 1.4816971 278R 1.2602246 1.3802029 281A 1.0778757 1.302493 281L 1.0664433 1.5428781 281M 1.2357293 1.317267 302D 1.3067743 1.3085968 302M 1.0807557 1.2463993 304D 1.1358974 1.304862 304E 1.1872403 1.2138013 304M 1.2316987 1.2224245 321A 1.0826055 1.2311805 321H 1.384587 1.4691649 321Q 1.3306703 1.3485614 321R 1.2446359 1.3138378 333Q 1.425789 1.6656427 378D 1.0880667 1.2202146 378N 1.0064817 1.2616767 378R 1.0264777 1.2826859 382D 1.1628676 1.2206133 398A 1.0127464 1.2067063 418A 1.070915 1.3701437 418M 1.101424 1.3091549 418N 1.1440828 1.4650527 420A 1.1288416 1.2216203 421R 1.142674 1.2396538 432A 1.1828859 1.4534375 432D 1.1261465 1.2701694 432L 1.1040571 1.2896033 432M 1.1530369 1.3947422 432N 1.1373288 1.2843802 432Q 1.2305257 1.3438957 432R 1.1226193 1.2108348 432Y 1.0242088 1.2209025 437D 1.0648095 1.2263069 437G 1.1270339 1.2057266 437H 1.0624587 1.2128077 437L 1.0706178 1.2702869 437M 1.1727007 1.3357945 437Y 1.2190374 1.428939 446A 1.2002798 1.498028 446Y 1.1205486 1.2525673 454A 0.9816646 1.2570919 464Q 1.0719588 1.2199585 464Y 1.1888873 1.3747167 474A 1.1556971 1.3935021 474E 1.1729152 1.3481142 474K 1.1240341 1.2036886 474L 1.110407 1.267509 474M 1.1869843 1.3422689 474N 1.1135684 1.2124349 474P 1.0761861 1.2293237 474Q 1.2580448 1.3477339 474R 1.1994238 1.3506214 474S 1.2348915 1.2615358 474V 1.0823992 1.2078523

TABLE 24-3 Positions in AmyS protein with mutations (shown in column labeled variant) that have performance indices for activity or expression at least 20% greater than wildtype AmyS Residual Variant Activity Activity 124N 1.2073767 1.1957849 125A 1.372718 −0.3461869 125K 1.2754087 −0.3195654 125N 1.2111343 1.111989 130A 1.2829276 −0.1606582 130S 1.2547959 −0.2396474 159A 1.2514424 1.8489959 159D 1.3042895 1.616517 159E 1.3048703 1.6426287 159G 1.309088 1.4806394 159H 1.2239861 1.4766606 159K 1.3024788 1.6188749 159L 1.2438467 1.7685564 159N 1.4021695 1.747298 159R 1.3445318 1.6062932 159S 1.3352659 1.5322275 159T 1.2115923 1.5982316 166F 1.3226117 0.9751853 166G 1.3251188 −0.8989095 166H 1.5608888 0.889625 166S 1.5553953 −0.4698927 166Y 1.3161377 0.9404254 169L 1.2709 1.221157 179A 1.2856782 1.4150905 179P 1.2367832 −0.2832651 180A 1.223674 1.1463487 180D 1.3732003 0.5446904 180H 1.3854073 −0.9190277 180K 1.4038831 −1.1078033 180L 1.6414819 −0.6936105 180N 1.2646998 −1.0108408 180T 1.4553893 −0.8759486 180V 1.2190216 −1.0611484 180Y 1.3113267 0.6162484 181A 1.5853606 2.5498838 181D 1.2451756 1.4958763 181E 1.2126846 1.3673333 181G 1.2893058 0.9117403 181P 1.3407541 1.8191875 181R 2.1023852 1.000651 181S 1.2285225 0.9373869 181V 1.2072805 1.2882775 187A 1.3658382 −0.221251 187C 1.3181513 −0.2335241 187K 1.2523832 −0.2685104 187N 1.2632558 0.127576 187P 1.4102122 −0.2495879 187Q 1.2477941 −0.2008265 187R 1.3445711 −0.2482154 187S 1.2513011 −0.2208563 242H 1.280464 0.7629545 242N 1.29758 0.8729278 278H 1.2287582 1.3214257 278K 1.2908668 1.3351968 278N 1.2587781 1.4816971 278R 1.2602246 1.3802029 281M 1.2357293 1.317267 302D 1.3067743 1.3085968 304M 1.2316987 1.2224245 304Y 1.3289811 1.1964204 321H 1.384587 1.4691649 321Q 1.3306703 1.3485614 321R 1.2446359 1.3138378 333Q 1.425789 1.6656427 432Q 1.2305257 1.3438957 437Y 1.2190374 1.428939 446A 1.2002798 1.498028 474Q 1.2580448 1.3477339 474S 1.2348915 1.2615358

Based on the relative performance data and stability data for the AmyS positions described in Table 23-1, 24-1, 24-2, and Table 24-3, AmyS positions were classified as restrictive versus non-restrictive as follows: Non-restrictive positions have>20% neutral mutations for at least one property. These positions are good candidates for mutation when making engineered α-amylases because mutations at these position have a high probability of improving performance. Restrictive positions have<20% neutral mutations for activity and stability. These positioned are generally left alone (i.e., not mutated) when engineering α-amylase variants, as mutation at these positions tend to reduce, rather than increase performance. All the positions/sites described in Table 23-1 are non-restrictive.

Example 25 Additional Positional Libraries in AmyS Protein

In addition to the AmyS variants described in Example 22, positional libraries at additional sites were generated in G. stearothermophilus α-amylase with a truncation (SEQ ID NO: 2). The libraries were produced by Geneart (Geneart GmbH, Josef-Engert-strasse 11, D-93053 Regensburg, Del.). Table 25-1 shows the site variants that were generated:

TABLE 25-1 Site variants generated in AmyS N5: A, C, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, Y G6: A, D, E, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y E13: A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y W14: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, Y Y15: A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, W L16: A, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, Y D18: A, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y G20: A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y K25: A, C, D, E, F, G, H, L, M, N, P, Q, R, S, T, Y A27: C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y E29: A, D, G, H, K, L, M, N, P, Q, R, S, T, W, Y L36: A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, Y T39: C, D, E, F, G, H, K, M, N, P, Q, R, S, V, W T50: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, Y R52: A, C, D, E, G, H, K, L, M, N, P, Q, S, T, V, W, Y S53: A, C, D, E, F, G, H, I, K, L, M, P, Q, R, T, V, W, Y D54: A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y E67: A, C, D, G, H, K, L, M, N, P, Q, R, S, T, W, Y K71: A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, Y T73: A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, V, W, Y R75: A, C, D, E, F, G, H, I, L, M, P, Q, S, T, V, W, Y K77: A, C, D, E, F, G, I, L, M, N, P, Q, R, S, T, V, W T80: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, Y K81: A, C, D, E, G, H, I, L, M, N, P, Q, R, S, T, V, W, Y Q83: A, C, D, E, G, G, H, I, L, M, P, R, S, T, V, W, Y L85: A, C, D, E, G, H, I, K, M, N, P, Q, R, S, T, W, Y A90: C, D, E, F, G, H, I, K, L, M, N, O, Q, R, S, T, V, W, Y H92: C, D, E, F, G, K, L, N, P, Q, R, S, T, V, W, Y H106: A, C, D, E, G, I, K, L, N, P, Q, R, S, T, V, W, Y K107: A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, Y D111: A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y T113: A, C, D, E, F, G, H, I, K, L, M, P, Q, R, V, W E114: A, C, D, F, G, H, I, L, M, N, P, R, T, V, W, Y E120: A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, Y V121: A, C, D, E, F, G, H, I, L, M, P, Q, R, S, T, W, Y R126: A, D, E, F, G, H, I, L, M, N, P, Q, T, V, W, Y Q128: A, C, D, E, G, H, I, K, L, N, P, R, S, T, V, W, Y S131: A, C, D, E, F, G, H, I, K, M, N, P, R, T, W, Y T133: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, Y Q137: A, C, D, E, F, G, H, I, L, M, P, R, S, T, V, W, Y A138: C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y W139: A, C, D, E, G, H, I, K, L, M, N, Q, R, S, T, V, Y K141: A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, Y D143: A, C, E, G, H, I, K, L, M, N, P, T, V, W, Y R147: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, Y N149: A, D, E, F, G, H, I, K, L, M, Q, R, S, V, W T150: A, C, D, E, F, G, I, K, L, M, N, Q, R, S, V, Y Y151: A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W S152: A, C, D, E, F, G, H, I, K, M, N, Q, R, T, V, W, Y K155: A, C, D, E, G, H, L, M, N, P, Q, R, S, T, V, W, Y H160: A, C, D, E, F, G, I, L, M, N, P, Q, R, S, T, V, W, Y D165: A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y E168: A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y L172: A, C, D, E, G, H, I, K, M, N, P, Q, R, S, T, V, W, Y S173: A, C, D, E, F, G, H, I, K, L, M, N, Q, R, T, V, W, Y K177: A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, W, Y E188: A, C, D, F, G, H, I, K, M, N, P, Q, S, T, V, W, Y T191: A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, V, W E192: A, C, D, G, H, I, K, M, N, P, Q, R, S, T, V, W, Y N193: A, C, D, E, F, G, H, I, K, L, M, P, R, S, T, W, Y Y196: A, C, D, E, F, G, H, I, K, L, N, P, R, S, T, V, W L199: A, E, G, H, I, K, M, N, P, Q, R, S, T, V, W, Y M200: A, C, D, E, F, G, H, I, K, L, N, P, Q, S, T, V, W Y201: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W A202: C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, Y T213: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W K216: A, D, E, F, G, H, I, L, M, P, Q, R, S, T, V, W, Y N217: A, C, E, F, G, H, I, L, M, P, Q, R, S, T, V, W, Y K220: A, C, D, E, F, G, H, I, M, N, P, Q, R, S, T, V, W, Y W221: A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, V, Y N227: A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, Y R232: A, C, D, E, G, H, K, M, N, P, Q, S, T, V, W, Y A235: C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y K237: A, C, D, E, F, G, H, I, L, M, N, P, Q, R, T, V, W, Y H238: A, C, D, F, G, I, K, L, M, N, P, Q, R, T, V, Y K240: A, D, E, F, G, H, I, M, N, P, Q, R, S, T, V, W, Y D246: A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, Y S249: A, C, D, E, F, G, H, K, L, M, P, Q, R, T, V, W, Y Y250: A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W R252: A, C, D, E, F, G, I, K, L, M, N, P, Q, S, T, V, Y S253: A, D, E, F, G, H, I, K, L, M, N, P, Q, T, V, W, Y Q254: A, C, D, E, F, G, H, I, K, L, M, N, R, S, T, V, W, Y T255: A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, V, W, Y K257: A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W P258: A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y Y268: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W K272: A, C, D, E, F, G, H, I, M, N, P, R, S, T, V, W, Y H274: A, C, D, E, F, G, I, K, L, N, P, Q, R, S, T, W, Y N275: A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, Y K279: C, E, F, G, H, I, L, M, N, P, Q, S, T, V, W, Y T283: A, C, D, E, G, H, I, K, L, M, N, P, R, S, V, W, Y S285: A, C, D, E, F, H, I, K, L, M, Q, R, T, V, W, Y N293: A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, Y K294: A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, Y T297: C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, Y K300: A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W S301: A, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, Y D306: A, C, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y T309: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, v, W, Y T312: A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, V, W, Y N313: A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, V, W K317: A, C, D, E, F, G, L, M, N, P, Q, R, S, T, V, W, Y D318: A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y Q319: A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, Y P320: A, C, D, E, F, G, H, I, K, L, M, Q, R, S, T, V, W, Y L338: A, C, D, E, F, G, H, I, K, M, P, Q, R, S, T, V, W, Y Q339: A, C, D, E, F, G, H, K, L, M, P, R, S, T, V, W, Y S340: A, C, D, E, F, H, I, K, L, M, N, P, Q, T, V, Y D343: A, C, E, F, H, I, L, M, N, P, Q, R, T, W, Y W345: A, C, D, E, F, H, I, K, L, M, N, P, Q, S, T, V C363: A, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, Y Y366: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W Y369: A, C, E, F, G, H, I, K, M, P, Q, R, S, T, V, W Y370: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W Y375: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W S379: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, Y K381: A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, Y D385: A, C, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W P386: A, C, D, E, F, G, H, I, K, L, M, N, Q, S, T, V, W, Y R391: A, C, E, G, H, K, L, N, P, Q, S, T, V, W, Y R392: A, C, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, Y D393: A, C, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y Y394: A, D, E, F, G, H, I, K, L, M, N, P, Q, S, V, W H400: A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, Y Y402: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W L403: A, C, D, E, F, G, H, M, N, P, Q, R, S, T, V, W, Y D404: A, C, E, G, I, K, L, M, N, P, Q, R, S, V, W, Y S406: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, T, V, Y D407: C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y G410: A, C, D, E, F, H, I, L, M, N, P, Q, R, T, V, W, Y R413: A, D, E, G, H, I, K, L, M, N, P, Q, S, T, V, W, Y E414: A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, Y V416: A, C, D, F, H, I, K, L, M, N, P, Q, R, S, T, W, Y K419: A, C, D, E, F, H, I, L, M, N, P, Q, R, S, T, V, W, Y S422: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, Y L427: A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, Y G433: A, C, D, E, F, H, I, K, L, M, N, P, W, R, S, T, V, Y K436: A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, Y Y439: A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W K442: A, C, F, G, H, I, N, P, Q, R, S, T, V, W, Y A445: C, D, G, H, I, K, L, N, P, Q, R, S, T, V, W K447: A, C, D, F, G, H, I, L, M, N, Q, R, S, T, V, W, Y V448: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, Y Y450: A, C, D, E, G, H, K, L, M, N, P, Q, R, S, T, V, W L452: A, C, D, E, F, G, H, K, M, N, P, Q, R, S, T, V, Y N455: A, C, D, E, G, H, I, K, L, M, P, Q, R, S, T, V, W, Y N463: A, D, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, Y D465: A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y E469: A, C, D, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, Y K471: A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, Y N473: A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, Y S476: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, Y

Example 26 Altered Properties of Variants Described in Example 25

This example shows that G. stearothermophilus alpha-amylase (AmyS) variants (described in Example 25) may have an altered property relative to the parent α-amylase. A high throughput thermal stability screen of AmyS variants was carried out as described in Example 3. Performance indices for activity (measured as BODIPY assay) and residual activity (after thermal stress) are shown in Tables 26-1, 26-2, 26-3.

TABLE 26-1 Positions in AmyS protein with mutations (shown in column labeled variant) that have performance indices for both activity and residual activity after heat stress better than wildtype AmyS. Residual Variant Activity Activity 006A 1.59 1.10 006D 1.64 1.14 006E 1.93 1.08 006I 1.47 1.23 006L 1.61 1.15 006M 1.60 1.11 006N 1.61 1.21 006P 2.47 1.10 006Q 1.34 1.26 006S 1.86 1.12 006T 2.01 1.21 006V 1.54 1.29 006W 1.32 1.13 006Y 1.88 1.07 014F 1.25 1.06 014T 1.22 1.22 014Y 1.71 1.08 015A 1.48 1.05 015H 1.85 1.01 016E 1.21 1.09 025C 1.46 1.33 025D 1.03 1.06 025H 1.06 1.03 025Q 1.07 1.24 027N 1.00 1.06 036K 1.05 1.01 036M 1.05 1.05 039C 1.05 1.09 039D 1.47 1.15 039E 1.32 1.15 039G 1.05 1.23 039H 1.10 1.16 039K 1.10 1.12 039N 1.64 1.14 039Q 1.43 1.20 039R 1.10 1.01 039S 1.02 1.15 050G 1.18 1.00 050N 1.12 1.20 050Q 1.08 1.31 050S 1.09 1.07 052M 1.00 1.01 052T 1.00 1.11 053A 1.00 1.03 053H 1.00 1.12 053K 1.10 1.03 053T 1.02 1.25 067G 1.13 1.01 067H 1.03 1.04 071R 1.10 1.10 075A 1.14 1.05 075M 1.04 1.04 085E 1.02 1.09 085M 1.04 1.01 085S 1.04 1.02 090H 1.03 1.05 090M 1.02 1.02 113L 1.08 1.08 133P 1.08 1.41 138P 1.15 1.07 138S 1.02 1.12 138T 1.05 1.16 139Y 1.63 1.14 141M 1.01 1.23 141N 1.02 1.11 143G 1.09 1.13 143V 1.01 1.00 150M 1.00 1.05 160N 1.02 1.11 165N 1.10 1.16 172A 1.06 1.14 172R 1.06 1.16 173K 1.03 1.17 188P 1.16 1.40 193K 1.17 1.28 193Y 1.10 1.89 201H 1.44 1.06 201M 1.21 1.16 213Q 1.02 1.12 213R 1.05 1.05 213S 1.08 1.10 216E 1.30 1.03 216Q 1.34 1.04 221F 1.28 1.07 221I 1.23 1.33 221M 1.35 1.16 221N 1.57 1.11 221S 1.40 1.34 221V 1.31 1.13 221Y 1.36 1.14 227A 1.02 1.01 227D 1.06 1.01 227E 1.06 1.03 227G 1.09 1.05 227K 1.13 1.00 235R 1.14 1.03 246E 1.03 1.18 249K 1.02 1.15 249R 1.03 1.07 250C 1.12 1.03 250E 1.33 1.13 250F 1.28 1.29 250G 1.33 1.09 250I 1.27 1.35 250K 1.48 1.07 250L 1.32 1.02 250M 1.39 1.35 250N 1.40 1.05 250Q 1.54 1.01 250S 1.41 1.02 252A 1.08 1.12 252E 1.12 1.09 252K 1.21 1.19 252Q 1.04 1.16 252S 1.01 1.04 253D 1.04 1.07 253K 1.01 1.10 253N 1.03 1.06 258D 1.10 1.33 258G 1.02 1.30 258H 1.13 1.38 258K 1.11 1.29 258N 1.01 1.07 258Q 1.13 1.31 258R 1.13 1.02 258S 1.08 1.12 258T 1.10 1.27 258Y 1.08 1.16 268F 1.07 1.28 268G 1.21 1.03 268S 1.22 1.06 274Y 1.07 1.05 283K 1.01 1.14 283S 1.06 1.02 283Y 1.04 1.01 285F 1.02 1.18 285Q 1.22 1.38 285W 1.08 1.13 293H 1.05 1.12 293K 1.41 1.42 293Q 1.06 1.14 293T 1.12 1.10 297R 1.14 1.03 301G 1.05 1.02 301K 1.05 1.08 309K 1.08 1.18 309R 1.08 1.12 312A 1.00 1.01 312G 1.07 1.18 313R 1.13 1.19 313S 1.05 1.25 318H 1.10 1.12 318S 1.37 1.11 318T 1.32 1.40 318Y 1.33 1.10 319A 1.13 1.02 319G 1.03 1.14 319K 1.52 1.10 319R 1.44 1.18 319V 1.08 1.07 319W 1.08 1.05 319Y 1.41 1.04 320S 1.03 1.16 320T 1.28 1.11 320Y 1.03 1.05 338A 1.29 1.36 338G 1.34 1.38 338I 1.32 1.12 338M 1.27 1.20 338P 1.23 1.11 338S 1.51 1.13 338T 1.05 1.42 338V 1.55 1.14 339A 1.13 1.08 339G 1.21 1.17 339H 1.04 1.03 339K 1.13 1.26 339P 1.24 1.02 339S 1.02 1.02 339T 1.01 1.35 340A 1.43 1.23 340H 1.45 1.12 340I 1.07 1.07 340M 1.20 1.24 340N 1.75 1.10 340Q 1.76 1.21 340T 1.14 1.21 343E 1.07 1.00 343P 1.03 1.30 343Q 1.01 1.14 343R 1.03 1.25 345D 1.15 1.10 345E 1.24 1.06 345H 1.10 1.15 345M 1.01 1.02 345N 1.10 1.07 345Q 1.10 1.26 345S 1.12 1.01 345T 1.15 1.15 345V 1.02 1.16 366H 1.12 1.07 366Q 1.49 1.03 366S 1.02 1.07 369M 1.02 1.06 370A 1.21 1.03 370G 1.18 1.21 370N 1.41 1.04 370S 1.50 1.06 370T 1.10 1.07 370V 1.13 1.05 375A 1.39 1.03 375L 1.07 1.03 375T 1.04 1.25 379A 1.02 1.01 385Q 1.01 1.02 392K 1.09 1.10 394K 1.07 1.09 394L 1.11 1.22 394Q 1.13 1.09 394S 1.15 1.11 394W 1.16 1.11 402T 1.02 1.32 403R 1.01 1.36 403V 1.00 1.34 413A 1.06 1.02 419A 1.29 1.36 419I 1.32 1.12 419M 1.27 1.20 419P 1.23 1.11 419S 1.51 1.13 419T 1.05 1.42 419V 1.55 1.14 422N 1.03 1.12 433A 1.08 1.27 433K 1.05 1.27 433M 1.01 1.23 433Y 1.01 1.26 442G 1.02 1.23 442H 1.04 1.07 442N 1.03 1.39 442P 1.03 1.11 442Q 1.05 1.11 442R 1.01 1.33 442S 1.07 1.24 442T 1.06 1.34 442Y 1.08 1.24 445G 1.01 1.21 447A 1.06 1.09 447L 1.01 1.06 448D 1.02 1.15 448F 1.01 1.48 448G 1.05 1.26 448H 1.03 1.37 448K 1.07 1.20 448L 1.08 1.04 448Q 1.16 1.18 448S 1.10 1.20 448Y 1.27 1.33 450R 1.02 1.22 450S 1.01 1.22 452A 1.06 1.08 452G 1.00 1.07 452K 1.08 1.11 452M 1.09 1.13 452N 1.28 1.06 452T 1.18 1.02 452V 1.14 1.14 452Y 1.07 1.17 455A 1.04 1.07 455G 1.00 1.23 455H 1.01 1.05 455K 1.08 1.10 455R 1.02 1.13 463A 1.06 1.25 463G 1.00 1.04 463L 1.01 1.16 463M 1.08 1.24 469A 1.01 1.16 469D 1.02 1.22 469F 1.00 1.11 469Q 1.04 1.03 469T 1.06 1.15 469V 1.08 1.15 469Y 1.09 1.35 471A 1.09 1.09 471D 1.06 1.01 471F 1.05 1.10 471G 1.12 1.13 471I 1.02 1.22 471N 1.12 1.04 471T 1.09 1.11 471V 1.11 1.28 471Y 1.36 1.15 473K 1.02 1.02 473M 1.00 1.11 473R 1.05 1.08 473T 1.04 1.04 476A 1.02 1.51 476M 1.08 1.58 476Q 1.03 1.13 476R 1.08 1.01 476T 1.01 1.78

TABLE 26-2 Positions in AmyS protein with mutations (shown in column labeled variant) that have performance indices for residual activity after heat stress at least 20% better than wildtype AmyS and performance indices for starting activity or expression at least half of wildtype AmyS. Residual Variant Activity Activity 006I 1.47 1.23 006N 1.61 1.21 006Q 1.34 1.26 006T 2.01 1.21 006V 1.54 1.29 014T 1.22 1.22 016F 0.98 2.17 025A 0.82 1.22 025C 1.46 1.33 025G 0.97 1.27 025Q 1.07 1.24 027M 0.88 1.21 036Q 0.78 1.40 036S 0.69 1.25 039G 1.05 1.23 039V 0.82 1.30 050I 0.61 1.24 050L 0.67 1.22 050M 0.62 1.32 050N 1.12 1.20 050Q 1.08 1.31 052S 0.92 1.21 053T 1.02 1.25 067N 0.95 1.32 067S 1.00 1.23 080D 0.86 1.22 080I 0.82 1.29 090E 0.92 1.20 133P 1.08 1.41 133V 0.92 1.25 137M 0.83 1.30 137S 0.98 1.45 141E 0.92 3.48 141I 0.87 1.40 141L 0.85 1.22 141M 1.01 1.23 141Q 0.97 1.28 141R 0.99 1.23 141S 0.98 1.21 141V 1.00 1.21 150E 0.87 4.54 151I 0.78 1.22 152G 0.91 1.25 155S 0.85 1.22 155Y 0.80 1.21 168W 0.66 1.23 173T 0.86 1.33 188P 1.16 1.40 193F 0.98 1.71 193K 1.17 1.28 193L 0.78 1.22 193Y 1.10 1.89 213L 0.75 1.26 213M 0.78 1.26 213V 0.76 1.35 217Q 0.74 1.31 220P 0.89 1.33 220Q 0.87 1.21 220R 0.83 1.26 220S 0.81 1.30 220V 0.82 1.21 221I 1.23 1.33 221S 1.40 1.34 249E 0.92 1.27 250F 1.28 1.29 250I 1.27 1.35 250M 1.39 1.35 252L 0.96 1.32 253Y 0.94 1.34 254E 0.89 1.29 254F 0.74 1.23 254T 0.65 1.21 254V 0.92 1.31 255F 0.68 1.30 255K 0.83 1.27 255W 0.74 1.27 257L 0.74 1.26 257M 0.79 1.29 257S 0.71 1.29 257V 0.78 1.31 258D 1.10 1.33 258G 1.02 1.30 258H 1.13 1.38 258K 1.11 1.29 258Q 1.13 1.31 258T 1.10 1.27 258V 0.80 1.29 268F 1.07 1.28 274W 0.79 1.26 283M 0.93 1.26 283N 0.96 1.29 283V 0.81 1.23 285E 0.89 1.33 285Q 1.22 1.38 293G 0.92 1.31 293K 1.41 1.42 294W 0.66 1.23 301F 0.68 1.44 301I 0.74 1.28 301P 0.61 1.21 301R 0.89 1.35 301T 0.85 1.23 301W 0.75 1.27 309D 0.89 1.27 309V 0.95 1.38 312H 0.99 1.30 312S 0.99 1.29 312V 0.87 1.40 312Y 0.92 1.31 313G 0.96 1.25 313H 0.94 1.25 313I 0.55 1.44 313L 0.85 1.21 313S 1.05 1.25 313V 0.74 1.28 318T 1.32 1.40 338A 1.29 1.36 338C 0.67 1.24 338G 1.34 1.38 338M 1.27 1.20 338T 1.05 1.42 339K 1.13 1.26 339T 1.01 1.35 339V 0.76 1.23 340A 1.43 1.23 340M 1.20 1.24 340Q 1.76 1.21 340T 1.14 1.21 343C 0.74 1.32 343I 0.88 1.27 343P 1.03 1.30 343R 1.03 1.25 343Y 0.82 1.29 345I 0.90 1.28 345Q 1.10 1.26 369I 0.91 1.33 369T 0.68 1.28 370G 1.18 1.21 375T 1.04 1.25 385T 0.92 1.22 386K 0.87 1.22 394L 1.11 1.22 394V 0.75 3.00 400A 0.89 1.24 400N 0.92 1.26 400V 0.91 1.28 402H 0.91 1.21 402I 0.75 1.36 402T 1.02 1.32 402V 0.95 1.40 402W 0.89 1.24 403A 0.89 1.20 403E 0.93 1.26 403G 0.96 1.22 403Q 0.98 1.24 403R 1.01 1.36 403T 0.99 1.53 403V 1.00 1.34 404C 0.61 1.28 404E 0.78 1.38 404G 0.77 1.25 404I 0.84 1.20 404V 0.79 1.28 419A 1.29 1.36 419C 0.67 1.24 419M 1.27 1.20 419T 1.05 1.42 422E 0.78 1.31 422G 0.99 1.20 433A 1.08 1.27 433H 0.99 1.27 433I 0.86 1.37 433K 1.05 1.27 433L 0.90 1.30 433M 1.01 1.23 433V 0.95 1.27 433Y 1.01 1.26 442A 0.98 1.38 442G 1.02 1.23 442N 1.03 1.39 442R 1.01 1.33 442S 1.07 1.24 442T 1.06 1.34 442V 0.99 1.20 442W 0.98 1.32 442Y 1.08 1.24 445G 1.01 1.21 445I 0.84 1.25 445N 0.91 1.20 445T 0.88 1.29 445V 0.93 1.27 445W 0.80 1.25 447I 0.91 1.22 447N 0.97 1.43 447Q 1.00 1.34 447W 0.89 1.31 447Y 0.96 1.21 448C 0.98 1.36 448F 1.01 1.48 448G 1.05 1.26 448H 1.03 1.37 448I 0.97 1.44 448N 0.70 1.24 448Y 1.27 1.33 450C 0.84 1.22 450H 0.90 1.23 450M 0.89 1.29 450N 0.96 1.23 450R 1.02 1.22 450S 1.01 1.22 450T 0.96 1.32 450W 0.95 1.21 455G 1.00 1.23 455I 0.95 1.23 455P 0.93 1.36 455V 0.89 1.26 463A 1.06 1.25 463M 1.08 1.24 463S 0.96 1.27 463T 0.91 1.38 463V 0.86 1.32 463W 0.74 1.45 465G 0.92 1.35 465I 0.85 1.37 465K 0.88 1.53 465N 0.93 1.32 465T 0.92 1.42 465V 0.93 1.24 469D 1.02 1.22 469W 0.97 1.24 469Y 1.09 1.35 471I 1.02 1.22 471V 1.11 1.28 473G 0.99 1.35 473Y 0.86 1.23 476A 1.02 1.51 476G 0.97 1.22 476L 0.93 1.46 476M 1.08 1.58 476N 0.98 1.61 476T 1.01 1.78

TABLE 26-3 Positions in AmyS protein with mutations (shown in column labeled variant) that have performance indices for activity or expression at least 20% greater than wildtype AmyS Variant Activity 006A 1.59 006D 1.64 006E 1.93 006H 2.29 006I 1.47 006K 2.36 006L 1.61 006M 1.60 006N 1.61 006P 2.47 006Q 1.34 006R 1.28 006S 1.86 006T 2.01 006V 1.54 006W 1.32 006Y 1.88 013K 1.22 014F 1.25 014T 1.22 014Y 1.71 015A 1.48 015D 1.82 015E 1.96 015G 1.89 015H 1.85 015K 1.58 015N 1.88 015P 1.59 015Q 1.74 015R 1.60 015S 1.78 015T 1.47 015W 1.44 016A 1.31 016E 1.21 016G 1.35 016H 1.21 016K 1.41 016N 1.32 016P 1.30 016Q 1.33 016R 1.28 016T 1.32 025C 1.46 039D 1.47 039E 1.32 039N 1.64 039Q 1.43 081Y 1.20 121P 1.22 139D 1.40 139H 1.59 139R 1.29 139Y 1.63 177A 1.20 188D 1.21 191H 1.27 191K 1.33 192A 1.26 192D 1.50 192G 1.38 192N 1.35 192P 1.33 192Q 1.55 192S 1.47 192T 1.35 192V 1.25 192Y 1.30 196A 1.57 196C 1.36 196D 1.29 196E 1.29 196F 1.38 196H 1.92 196I 1.61 196K 1.29 196P 1.50 196R 1.29 196S 1.59 196T 1.65 196V 1.55 201A 1.41 201E 1.36 201G 1.63 201H 1.44 201M 1.21 202H 1.30 216E 1.30 216G 1.20 216H 1.28 216M 1.39 216Q 1.34 216R 1.32 216S 1.28 216T 1.22 216Y 1.31 221A 1.54 221D 1.31 221F 1.28 221I 1.23 221L 1.50 221M 1.35 221N 1.57 221R 1.29 221S 1.40 221V 1.31 221Y 1.36 237G 1.21 240G 1.22 240N 1.37 240P 1.69 240Q 1.21 240R 1.41 240T 1.23 246R 1.31 250A 1.21 250D 1.29 250E 1.33 250F 1.28 250G 1.33 250I 1.27 250K 1.48 250L 1.32 250M 1.39 250N 1.40 250Q 1.54 250R 1.55 250S 1.41 250W 1.35 252K 1.21 268A 1.39 268D 1.44 268E 1.47 268G 1.21 268H 1.24 268K 1.90 268N 1.51 268P 1.41 268Q 1.30 268R 1.49 268S 1.22 274A 1.40 274D 1.20 274G 1.36 274I 1.39 274K 1.60 274L 1.40 274N 1.50 274Q 1.47 274R 1.50 274S 1.28 274T 1.38 275K 1.22 285Q 1.22 285Y 1.49 293K 1.41 293R 1.37 318A 1.38 318F 1.22 318G 1.39 318I 1.40 318K 1.73 318L 1.31 318M 1.26 318R 1.54 318S 1.37 318T 1.32 318V 1.34 318Y 1.33 319C 1.38 319D 1.31 319H 1.28 319I 1.32 319K 1.52 319R 1.44 319Y 1.41 320K 1.23 320R 1.25 320T 1.28 338A 1.29 338G 1.34 338I 1.32 338M 1.27 338P 1.23 338S 1.51 338V 1.55 339G 1.21 339P 1.24 340A 1.43 340D 1.63 340E 1.58 340H 1.45 340K 1.76 340N 1.75 340Q 1.76 345E 1.24 363D 1.74 363E 1.34 363M 1.36 363N 1.86 363Q 1.78 363S 1.35 366Q 1.49 370A 1.21 370D 1.35 370E 1.35 370H 1.36 370K 1.65 370N 1.41 370Q 1.51 370S 1.50 375A 1.39 375D 1.52 375E 1.48 375K 1.43 375N 1.48 375Q 1.56 375R 1.61 375S 1.29 419A 1.29 419I 1.32 419M 1.27 419P 1.23 419S 1.51 419V 1.55 448Y 1.27 452N 1.28 452Q 1.22 452R 1.26 452S 1.21 471R 1.33 471Y 1.36

Table 26-4 shows the Performance index values (Pi) for 2,666 variants of AmyS at 152 positions. Performance indices less than or equal to 0.05 in the activity assay were fixed to 0.05 and indicated in bold italics in the Table 26-4. Also, for the stability measure, if the Performance index of activity in the stability assays was less than or equal to 0.05, the associated stability performance index was fixed to 0.05.

TABLE 26-4 Performance Indicies for stability and activity measurements for AmyS variants Stability Activity Position Variant PI PI 5 N005A 0.95 0.32 5 N005C 0.98 0.29 5 N005E 1.04 0.43 5 N005F 0.79 0.15 5 N005G 0.88 0.34 5 N005H 0.89 0.43 5 N005I 1.00 0.10 5 N005K 0.90 0.34 5 N005L 1.04 0.10 5 N005M 0.84 0.18 5 N005P 1.10 0.40 5 N005Q 1.07 0.58 5 N005R 0.94 0.40 5 N005S 0.98 0.35 5 N005T 0.83 0.35 5 N005V 0.88 0.16 5 N005W 0.94 0.07 5 N005Y 1.07 0.21 6 G006A 1.10 1.59 6 G006D 1.14 1.64 6 G006E 1.08 1.93 6 G006H 0.95 2.29 6 G006I 1.23 1.47 6 G006K 0.93 2.36 6 G006L 1.15 1.61 6 G006M 1.11 1.60 6 G006N 1.21 1.61 6 G006P 1.10 2.47 6 G006Q 1.26 1.34 6 G006R 0.98 1.28 6 G006S 1.12 1.86 6 G006T 1.21 2.01 6 G006V 1.29 1.54 6 G006W 1.13 1.32 6 G006Y 1.07 1.88 13 E013A 0.32 1.01 13 E013C 0.22 0.68 13 E013D 0.08 1.03 13 E013F

0.81 13 E013G 0.18 1.00 13 E013H 0.60 1.10 13 E013I 0.15 0.87 13 E013K 0.22 1.22 13 E013L 0.20 1.02 13 E013M 0.20 0.96 13 E013N

13 E013P

0.37 13 E013Q 0.21 0.96 13 E013R 0.28 1.04 13 E013S 0.28 0.92 13 E013T 0.19 0.79 13 E013V 0.19 0.76 13 E013W

0.76 13 E013Y 0.89 0.93 14 W014A 0.95 0.77 14 W014C 0.91 0.71 14 W014D 0.81 0.59 14 W014E 0.95 1.07 14 W014F 1.06 1.25 14 W014G 0.97 0.88 14 W014H

14 W014I 1.12 0.40 14 W014K 1.01 0.69 14 W014L 0.88 0.15 14 W014M 1.18 0.84 14 W014N 0.92 0.99 14 W014P 0.84 0.98 14 W014Q 0.94 0.67 14 W014R 0.97 0.67 14 W014S 0.97 1.02 14 W014T 1.22 1.22 14 W014V 1.17 0.81 14 W014Y 1.08 1.71 15 Y015A 1.05 1.48 15 Y015C 0.70 1.15 15 Y015D 0.77 1.82 15 Y015E 0.68 1.96 15 Y015G 0.69 1.89 15 Y015H 1.01 1.85 15 Y015I 0.63 0.91 15 Y015K 0.74 1.58 15 Y015L 0.67 0.76 15 Y015M 0.72 1.12 15 Y015N 0.99 1.88 15 Y015P 0.57 1.59 15 Y015Q 0.80 1.74 15 Y015R 0.72 1.60 15 Y015S 0.58 1.78 15 Y015T 0.87 1.47 15 Y015W 0.95 1.44 16 L016A 0.81 1.31 16 L016D 0.93 1.12 16 L016E 1.09 1.21 16 L016F 2.17 0.98 16 L016G 0.61 1.35 16 L016H 0.96 1.21 16 L016I 0.79 1.12 16 L016K 0.79 1.41 16 L016M 0.94 1.15 16 L016N 0.92 1.32 16 L016P 0.35 1.30 16 L016Q 0.96 1.33 16 L016R 0.71 1.28 16 L016S 0.94 1.19 16 L016T 0.87 1.32 16 L016V 0.87 1.16 16 L016W 0.75 0.99 16 L016Y 0.97 1.10 18 D018A 1.08 0.89 18 D018F 0.68 0.58 18 D018G 0.88 0.87 18 D018H 0.84 0.84 18 D018I 0.79 0.70 18 D018K 0.88 0.65 18 D018L 0.60 0.72 18 D018N 0.73 1.01 18 D018P 0.84 1.04 18 D018Q 0.80 1.00 18 D018R 0.81 0.65 18 D018S 0.81 0.93 18 D018T 0.81 0.91 18 D018V 0.89 0.77 18 D018W 0.72 0.51 18 D018Y 0.72 0.87 20 G020A 0.79 0.25 20 G020C 0.58 0.24 20 G020D 0.92 0.96 20 G020E 0.89 0.95 20 G020F 0.65 0.13 20 G020H 0.75 0.11 20 G020I 0.96 0.28 20 G020K

20 G020L

20 G020M 0.69 0.10 20 G020N 0.78 0.09 20 G020P

20 G020Q 0.61 0.07 20 G020R

20 G020S

20 G020T 0.82 0.09 20 G020V 0.77 0.19 20 G020W 0.80 0.69 20 G020Y

25 K025A 1.22 0.82 25 K025C 1.33 1.46 25 K025D 1.06 1.03 25 K025E 1.07 0.95 25 K025F 1.00 0.58 25 K025G 1.27 0.97 25 K025H 1.03 1.06 25 K025L 1.12 0.64 25 K025M 1.03 0.61 25 K025N 0.91 1.06 25 K025P 0.98 0.55 25 K025Q 1.24 1.07 25 K025R 1.08 0.96 25 K025S 1.07 0.98 25 K025T 1.14 0.89 25 K025Y 0.98 0.65 27 A027C 0.79 0.55 27 A027D 1.01 0.95 27 A027E 0.93 0.95 27 A027F 0.88 0.85 27 A027G 1.20 0.98 27 A027H 1.05 1.00 27 A027I 1.05 0.87 27 A027K 0.86 1.01 27 A027L 1.06 0.86 27 A027M 1.21 0.88 27 A027N 1.06 1.00 27 A027P 1.13 0.43 27 A027Q 1.00 0.96 27 A027R 1.11 0.89 27 A027S 1.16 0.97 27 A027T 1.20 0.90 27 A027V 1.20 0.82 27 A027W 1.13 0.76 27 A027Y 0.97 0.28 29 E029A 1.05 0.50 29 E029D 0.94 1.11 29 E029G 0.75 0.37 29 E029H 0.83 0.83 29 E029K 1.05 0.89 29 E029L 0.76 0.22 29 E029M 0.76 0.15 29 E029N 1.02 0.89 29 E029P 0.87 0.33 29 E029Q 1.04 0.86 29 E029R 1.09 0.92 29 E029S 0.97 0.83 29 E029T 0.95 0.59 29 E029W 0.74 0.10 29 E029Y

36 L036A 0.95 0.85 36 L036C 0.83 0.43 36 L036D 0.91 0.27 36 L036E 0.90 0.40 36 L036F 1.14 0.90 36 L036G 0.92 0.34 36 L036H 0.92 0.77 36 L036I 1.17 0.89 36 L036K 1.01 1.05 36 L036M 1.05 1.05 36 L036N 1.02 0.68 36 L036P 0.90 0.06 36 L036Q 1.40 0.78 36 L036R 1.12 0.76 36 L036S 1.25 0.69 36 L036T 1.11 0.64 36 L036V 0.88 0.97 36 L036W 0.92 0.63 36 L036Y 1.07 0.91 39 T039C 1.09 1.05 39 T039D 1.15 1.47 39 T039E 1.15 1.32 39 T039F 1.16 0.48 39 T039G 1.23 1.05 39 T039H 1.16 1.10 39 T039K 1.12 1.10 39 T039M 1.18 0.54 39 T039N 1.14 1.64 39 T039P 1.11 0.26 39 T039Q 1.20 1.43 39 T039R 1.01 1.10 39 T039S 1.15 1.02 39 T039V 1.30 0.82 39 T039W 1.11 0.25 50 T050A 1.09 0.98 50 T050C 1.03 0.34 50 T050D 0.87 0.91 50 T050E

50 T050F 0.86 0.43 50 T050G 1.00 1.18 50 T050H 0.97 0.82 50 T050I 1.24 0.61 50 T050K 1.13 0.80 50 T050L 1.22 0.67 50 T050M 1.32 0.62 50 T050N 1.20 1.12 50 T050P 1.03 0.99 50 T050Q 1.31 1.08 50 T050R 1.13 0.79 50 T050S 1.07 1.09 50 T050V 1.02 0.79 50 T050W 0.90 0.18 50 T050Y 1.14 0.42 52 R052A 0.99 1.02 52 R052C 0.87 0.62 52 R052D 0.76 0.85 52 R052E 0.77 0.97 52 R052G 0.96 0.93 52 R052H 0.91 0.99 52 R052K 0.93 1.02 52 R052L 1.10 0.98 52 R052M 1.01 1.00 52 R052N 0.95 0.99 52 R052P 1.05 0.95 52 R052Q

52 R052S 1.21 0.92 52 R052T 1.11 1.00 52 R052V 1.14 0.95 52 R052W 1.00 0.83 52 R052Y 0.99 0.96 53 S053A 1.03 1.00 53 S053C 0.73 0.58 53 S053D 0.75 0.83 53 S053E 1.05 0.88 53 S053F 0.87 0.85 53 S053G 1.14 0.93 53 S053H 1.12 1.00 53 S053I 0.99 1.12 53 S053K 1.03 1.10 53 S053L 0.93 0.96 53 S053M 0.96 0.97 53 S053P 0.88 1.00 53 S053Q 0.94 0.94 53 S053R 0.83 1.15 53 S053T 1.25 1.02 53 S053V 1.11 0.94 53 S053W 1.09 0.84 53 S053Y 0.94 0.93 54 D054A 0.34 0.88 54 D054C 0.64 0.38 54 D054E

54 D054F

0.60 54 D054G 0.11 0.97 54 D054H 0.11 1.04 54 D054I 0.30 0.83 54 D054K

1.08 54 D054L

0.89 54 D054M 0.11 0.88 54 D054N 0.94 1.05 54 D054P

1.03 54 D054Q

54 D054R 0.06 0.89 54 D054S 0.38 0.96 54 D054T 0.17 0.95 54 D054V 0.17 0.77 54 D054W

54 D054Y

0.64 67 E067A

67 E067C 1.08 0.75 67 E067D 0.90 1.07 67 E067G 1.01 1.13 67 E067H 1.04 1.03 67 E067K 0.98 0.94 67 E067L 0.97 0.95 67 E067M 0.93 0.91 67 E067N 1.32 0.95 67 E067P

67 E067Q 0.93 0.95 67 E067R 1.01 0.90 67 E067S 1.23 1.00 67 E067T 0.99 0.98 67 E067W

67 E067Y 1.11 0.93 71 K071A 0.72 0.81 71 K071C 0.80 0.61 71 K071D 0.69 0.71 71 K071E 0.80 0.84 71 K071F 0.47 0.61 71 K071G 0.74 0.91 71 K071H 0.96 0.88 71 K071I 0.83 0.75 71 K071L 0.55 0.61 71 K071M 0.80 0.68 71 K071N 1.11 0.89 71 K071P 0.92 0.86 71 K071Q 0.98 0.77 71 K071R 1.10 1.10 71 K071S 0.99 0.97 71 K071T 0.95 0.83 71 K071V 0.94 0.84 71 K071W 0.82 0.91 71 K071Y 0.52 0.71 73 T073A 0.97 1.11 73 T073C 0.91 0.60 73 T073D 0.89 1.02 73 T073E 0.75 1.08 73 T073F 0.73 0.99 73 T073G 0.79 1.12 73 T073H 0.86 0.88 73 T073I 0.66 1.02 73 T073K 0.20 0.97 73 T073L 0.47 1.17 73 T073M 0.59 0.64 73 T073N 0.73 1.08 73 T073P 0.57 0.98 73 T073R 0.40 1.11 73 T073S 0.87 1.10 73 T073V 0.67 1.09 73 T073W 0.83 1.07 73 T073Y 0.79 1.10 75 R075A 1.05 1.14 75 R075C 0.88 0.85 75 R075D 0.87 0.99 75 R075E 0.86 1.01 75 R075F 0.76 0.92 75 R075G 0.79 1.04 75 R075H 0.85 1.07 75 R075I 0.86 1.01 75 R075L 0.88 1.04 75 R075M 1.04 1.04 75 R075P 0.90 0.93 75 R075Q 0.90 0.95 75 R075S 0.66 0.60 75 R075T 0.98 0.88 75 R075V 0.78 0.94 75 R075W 0.75 0.93 75 R075Y 0.68 1.04 77 K077A 0.38 0.98 77 K077C 0.28 0.51 77 K077D

0.59 77 K077E 0.11 0.77 77 K077F 0.20 0.72 77 K077G 0.13 0.76 77 K077I 0.16 1.00 77 K077L 0.54 0.98 77 K077M 0.58 0.99 77 K077N

77 K077P

0.61 77 K077Q 0.07 0.86 77 K077R 0.77 1.07 77 K077S 0.11 0.89 77 K077T

0.86 77 K077V 0.05 0.83 77 K077W

0.77 80 T080A 0.88 1.01 80 T080C 0.91 0.69 80 T080D 1.22 0.86 80 T080E 0.71 0.92 80 T080F 1.10 0.50 80 T080G 1.02 0.93 80 T080H 1.01 0.95 80 T080I 1.29 0.82 80 T080K 0.90 0.86 80 T080L 0.82 0.98 80 T080M 0.97 0.95 80 T080N 0.90 1.00 80 T080P 0.88 0.88 80 T080Q 0.87 0.88 80 T080R 0.99 0.76 80 T080S 0.83 1.09 80 T080V 0.87 0.87 80 T080W 0.77 0.89 80 T080Y 0.72 0.97 81 K081A 0.87 0.94 81 K081C 0.84 0.74 81 K081D 0.96 0.83 81 K081E 0.69 0.92 81 K081G 0.86 0.81 81 K081H 0.73 1.03 81 K081I 0.82 0.79 81 K081L 0.87 1.01 81 K081M 0.93 1.04 81 K081N

81 K081P 0.90 0.79 81 K081Q 0.84 1.03 81 K081R 0.90 1.04 81 K081S 0.74 0.98 81 K081T 0.80 0.93 81 K081V 0.66 1.03 81 K081W 0.60 0.98 81 K081Y 0.89 1.20 83 Q083A 1.20 0.98 83 Q083C 1.79 0.17 83 Q083D 0.94 0.92 83 Q083E 0.98 0.95 83 Q083F 0.87 0.80 83 Q083G 0.76 1.01 83 Q083H 0.78 0.86 83 Q083I 0.69 0.85 83 Q083L 0.77 0.91 83 Q083M 0.91 0.96 83 Q083P 1.01 0.82 83 Q083R 0.91 0.90 83 Q083S 0.75 0.99 83 Q083T 0.84 0.84 83 Q083V 0.73 0.80 83 Q083W 0.82 0.78 83 Q083Y 0.71 0.93 85 L085A 0.94 1.06 85 L085C 0.90 0.63 85 L085D 0.84 1.04 85 L085E 1.09 1.02 85 L085G 0.85 0.90 85 L085H 0.73 1.02 85 L085I 0.89 0.88 85 L085K 0.96 0.93 85 L085M 1.01 1.04 85 L085N 1.10 0.89 85 L085P 1.01 0.72 85 L085Q 0.91 0.99 85 L085R 0.96 1.01 85 L085S 1.02 1.04 85 L085T 0.83 1.12 85 L085W 0.93 0.95 85 L085Y 0.70 1.08 90 A090C 1.00 0.65 90 A090D 1.12 0.92 90 A090E 1.20 0.92 90 A090F 0.99 0.76 90 A090G 1.04 0.87 90 A090H 1.05 1.03 90 A090I 0.90 0.83 90 A090K 0.93 1.04 90 A090L 0.76 0.92 90 A090M 1.02 1.02 90 A090N 1.02 0.98 90 A090P 1.39 0.10 90 A090Q 0.94 0.93 90 A090R 0.90 0.90 90 A090S 1.16 0.99 90 A090T 0.78 0.88 90 A090V 0.79 0.87 90 A090W 0.69 0.84 90 A090Y 0.83 0.96 92 H092C 0.75 0.29 92 H092D 1.06 0.69 92 H092E 0.88 0.76 92 H092F 0.92 0.28 92 H092G 0.86 0.81 92 H092K 0.89 0.98 92 H092L 0.43 0.12 92 H092N 0.85 0.78 92 H092P

92 H092Q 0.80 0.89 92 H092R 0.75 0.96 92 H092S 0.70 0.87 92 H092T 0.68 0.47 92 H092V 0.70 0.28 92 H092W 0.83 0.44 92 H092Y 0.71 0.63 106 H106A 0.32 0.19 106 H106C 0.33 0.06 106 H106D 0.58 0.07 106 H106E

106 H106G 0.16 0.17 106 H106I

106 H106K

106 H106L

0.06 106 H106N 0.14 0.08 106 H106P 0.59 0.06 106 H106Q 0.07 0.39 106 H106R

106 H106S

0.20 106 H106T

106 H106V

106 H106W

106 H106Y

107 K107A 0.46 0.81 107 K107C 0.42 0.67 107 K107D 0.32 0.51 107 K107E 0.35 0.70 107 K107F 0.42 0.66 107 K107G 0.23 0.76 107 K107H 0.34 0.94 107 K107I 0.29 0.69 107 K107L 0.53 0.75 107 K107M 0.60 0.79 107 K107N 0.43 0.88 107 K107P

0.65 107 K107Q 0.63 0.74 107 K107R 1.05 0.71 107 K107S 0.30 0.78 107 K107T 0.38 0.72 107 K107V 0.41 0.70 107 K107W

0.44 107 K107Y 0.40 0.64 111 D111A 0.55 0.95 111 D111C 0.71 0.60 111 D111E 0.87 1.01 111 D111F 0.63 0.65 111 D111G 0.74 0.90 111 D111H 0.50 0.85 111 D111I 0.56 0.91 111 D111K 0.45 0.62 111 D111L 0.44 0.86 111 D111M 0.65 1.00 111 D111N 0.97 0.87 111 D111P 0.78 0.71 111 D111Q 0.77 0.95 111 D111R 0.53 0.07 111 D111S 0.67 0.91 111 D111T 0.61 1.02 111 D111V 0.58 1.02 111 D111W 0.42 0.54 111 D111Y 0.49 0.92 113 T113A 0.89 0.97 113 T113C 0.80 0.82 113 T113D 0.94 0.95 113 T113E 0.92 0.91 113 T113F 0.76 0.92 113 T113G 0.88 1.08 113 T113H 0.88 0.96 113 T113I 1.14 0.88 113 T113K 0.93 1.13 113 T113L 1.08 1.08 113 T113M 0.83 0.99 113 T113P 1.05 0.96 113 T113Q 0.88 1.05 113 T113R 0.88 1.03 113 T113V 1.12 0.94 113 T113W 1.06 0.88 114 E114A 0.54 0.97 114 E114C 0.62 0.76 114 E114D 0.71 0.82 114 E114F 0.36 0.92 114 E114G 0.59 1.01 114 E114H 0.49 0.92 114 E114I 0.54 0.86 114 E114L 0.43 0.97 114 E114M 0.77 0.97 114 E114N 0.67 0.88 114 E114P 0.37 0.37 114 E114R 0.35 0.84 114 E114T 0.54 0.94 114 E114V 0.43 0.85 114 E114W 0.31 0.94 114 E114Y 0.26 0.93 120 E120A 0.29 1.20 120 E120C 0.24 0.89 120 E120D

1.02 120 E120F

0.88 120 E120G

1.14 120 E120H 0.09 0.90 120 E120I 0.60 0.87 120 E120L 0.20 0.97 120 E120M 0.39 0.96 120 E120N 0.16 1.02 120 E120P

1.12 120 E120Q 0.66 1.10 120 E120R 0.12 1.12 120 E120S 0.08 1.07 120 E120T 0.22 1.06 120 E120V 0.53 0.93 120 E120W 0.15 0.81 120 E120Y 0.07 0.98 121 V121A

1.04 121 V121C 0.92 0.55 121 V121D

0.91 121 V121E

0.93 121 V121F

0.77 121 V121G

0.92 121 V121H

121 V121I

0.79 121 V121L

0.98 121 V121M

0.97 121 V121P

1.22 121 V121Q

0.97 121 V121R

1.01 121 V121S

0.95 121 V121T 0.07 0.92 121 V121W

0.62 121 V121Y

0.88 126 R126A

126 R126D

0.46 126 R126E

0.82 126 R126F

1.03 126 R126G

0.89 126 R126H

1.06 126 R126I

0.95 126 R126L

0.97 126 R126M

1.01 126 R126N

1.07 126 R126P

0.67 126 R126Q

0.65 126 R126T

0.83 126 R126V

0.99 126 R126W

1.06 126 R126Y

1.01 128 Q128A

128 Q128C 0.42 0.95 128 Q128D 0.15 1.05 128 Q128E 0.90 1.00 128 Q128G

0.99 128 Q128H 0.34 1.05 128 Q128I 0.90 0.89 128 Q128K 0.52 1.15 128 Q128L 0.47 0.97 128 Q128N 0.12 1.05 128 Q128P

1.03 128 Q128R 0.31 1.14 128 Q128S 0.28 1.02 128 Q128T

128 Q128V 0.86 0.97 128 Q128W 0.07 0.76 128 Q128Y 0.13 0.86 131 S131A

1.15 131 S131C

0.98 131 S131D 0.26 1.08 131 S131E

1.14 131 S131F

0.92 131 S131G 0.24 0.86 131 S131H

1.13 131 S131I

131 S131K

1.13 131 S131M

0.99 131 S131N 0.76 1.02 131 S131P

1.05 131 S131R

1.05 131 S131T 0.49 0.90 131 S131W

0.82 131 S131Y

0.90 133 T133A 0.95 1.13 133 T133C 0.49 0.97 133 T133D 1.03 0.99 133 T133E 0.82 1.02 133 T133F 0.17 0.97 133 T133G 0.47 0.84 133 T133H 0.41 1.19 133 T133I 0.86 0.96 133 T133K 0.47 0.85 133 T133L 0.41 1.06 133 T133M 0.51 1.05 133 T133N 0.68 1.13 133 T133P 1.41 1.08 133 T133Q 0.63 1.10 133 T133R 0.18 1.13 133 T133S 0.72 1.08 133 T133V 1.25 0.92 133 T133W 0.14 0.98 133 T133Y 0.41 1.01 137 Q137A 0.92 0.97 137 Q137C 1.09 0.77 137 Q137D 0.89 0.96 137 Q137E 1.06 0.87 137 Q137F 0.85 0.86 137 Q137G 1.13 0.94 137 Q137H 0.95 1.05 137 Q137I 0.93 0.22 137 Q137L 1.20 0.82 137 Q137M 1.30 0.83 137 Q137P 0.07 1.05 137 Q137R 0.95 1.05 137 Q137S 1.45 0.98 137 Q137T 1.12 0.91 137 Q137V 1.02 0.86 137 Q137W 1.06 0.88 137 Q137Y 0.94 0.89 138 A138C

138 A138D

0.37 138 A138E

0.54 138 A138G 0.90 1.02 138 A138H

0.60 138 A138I 0.23 0.90 138 A138K

0.15 138 A138L

0.90 138 A138M

0.94 138 A138N 0.50 0.94 138 A138P 1.07 1.15 138 A138Q 0.13 0.69 138 A138R

0.15 138 A138S 1.12 1.02 138 A138T 1.16 1.05 138 A138V 1.17 0.87 138 A138W

0.27 138 A138Y 0.14 0.97 139 W139A 0.82 0.89 139 W139C 0.75 0.39 139 W139D 0.93 1.40 139 W139E 0.81 0.97 139 W139G 0.79 0.74 139 W139H 0.97 1.59 139 W139I 0.74 0.58 139 W139K 0.68 0.42 139 W139L 0.78 0.59 139 W139M 0.87 1.00 139 W139N 1.13 0.85 139 W139Q 0.82 0.79 139 W139R 0.96 1.29 139 W139S 0.93 1.04 139 W139T 0.71 0.87 139 W139V 0.72 0.66 139 W139Y 1.14 1.63 141 K141A 1.09 0.73 141 K141C 1.03 0.85 141 K141D 0.89 0.98 141 K141E 3.48 0.92 141 K141F 0.89 0.80 141 K141G 1.18 0.96 141 K141H 1.13 0.99 141 K141I 1.40 0.87 141 K141L 1.22 0.85 141 K141M 1.23 1.01 141 K141N 1.11 1.02 141 K141P 1.07 0.96 141 K141Q 1.28 0.97 141 K141R 1.23 0.99 141 K141S 1.21 0.98 141 K141T 1.17 0.94 141 K141V 1.21 1.00 141 K141W 1.16 0.87 141 K141Y 1.17 0.88 143 D143A 0.95 1.04 143 D143C 1.11 0.84 143 D143E 1.12 0.98 143 D143G 1.13 1.09 143 D143H 0.91 0.98 143 D143I 1.05 0.94 143 D143K 0.86 0.96 143 D143L

143 D143M 0.86 1.05 143 D143N 1.10 0.99 143 D143P 0.98 0.84 143 D143T

143 D143V 1.00 1.01 143 D143W 1.00 0.99 143 D143Y 0.75 0.15 147 R147A 0.73 0.25 147 R147C

147 R147D 0.66 0.07 147 R147E

147 R147F

147 R147G 0.74 0.11 147 R147H 0.81 0.21 147 R147I

147 R147K 1.05 0.48 147 R147L

147 R147M 0.65 0.07 147 R147N 0.91 0.30 147 R147P

147 R147Q 0.88 0.30 147 R147S 0.90 0.39 147 R147T 0.90 0.10 147 R147V

147 R147W

147 R147Y

149 N149A 0.94 0.93 149 N149D 0.89 0.95 149 N149E 0.98 0.93 149 N149F 1.09 0.85 149 N149G 0.90 0.93 149 N149H 1.01 0.98 149 N149I 1.15 0.83 149 N149K 0.90 0.88 149 N149L 0.88 0.94 149 N149M

149 N149Q 1.00 0.93 149 N149R 0.80 0.95 149 N149S 0.94 1.03 149 N149V 1.06 0.87 149 N149W 1.01 0.87 150 T150A 0.90 0.96 150 T150C 1.03 0.72 150 T150D 0.82 0.87 150 T150E 4.54 0.87 150 T150F

150 T150G 0.99 0.86 150 T150I 0.82 0.93 150 T150K 0.86 0.96 150 T150L 0.83 0.07 150 T150M 1.05 1.00 150 T150N 0.98 1.08 150 T150Q 0.83 0.99 150 T150R 0.99 1.04 150 T150S 0.77 0.96 150 T150V 0.90 0.93 150 T150Y 1.18 1.00 151 Y151A 0.96 0.87 151 Y151C 0.80 0.67 151 Y151D 0.99 0.71 151 Y151E 0.76 0.71 151 Y151F 0.96 0.88 151 Y151G 1.17 0.79 151 Y151H 1.04 0.87 151 Y151I 1.22 0.78 151 Y151L 1.05 0.90 151 Y151M 1.02 0.83 151 Y151N 0.98 0.91 151 Y151P 0.89 0.77 151 Y151Q 1.07 0.75 151 Y151R 1.05 0.76 151 Y151S 0.85 0.80 151 Y151T 1.04 0.80 151 Y151V 1.14 0.80 151 Y151W 1.16 0.79 152 S152A 0.95 0.88 152 S152C 0.83 0.75 152 S152D

152 S152E 1.09 0.71 152 S152F 0.75 0.22 152 S152G 1.25 0.91 152 S152H 0.99 0.71 152 S152I 0.81 0.22 152 S152K 0.74 0.58 152 S152M

152 S152N 1.20 0.43 152 S152Q 0.71 0.21 152 S152R 0.89 0.86 152 S152T 1.16 0.99 152 S152V 0.79 0.42 152 S152W 0.73 0.22 152 S152Y 0.91 0.26 155 K155A 1.10 0.85 155 K155C 0.92 0.72 155 K155D 0.94 0.85 155 K155E 0.82 0.79 155 K155G 1.05 0.58 155 K155H 1.04 0.84 155 K155L 1.05 0.89 155 K155M 0.91 0.91 155 K155N 1.18 0.90 155 K155P 0.99 0.94 155 K155Q 0.84 0.90 155 K155R 1.20 0.93 155 K155S 1.22 0.85 155 K155T 1.12 0.76 155 K155V 1.01 0.85 155 K155W 1.09 0.88 155 K155Y 1.21 0.80 160 H160A 0.89 0.89 160 H160C 0.84 0.98 160 H160D 0.89 0.69 160 H160E 0.86 0.52 160 H160F 0.77 0.79 160 H160G 0.82 0.36 160 H160I 0.36 0.58 160 H160L 1.03 0.92 160 H160M 0.56 0.97 160 H160N 1.11 1.02 160 H160P

160 H160Q 0.98 0.47 160 H160R 0.54 0.62 160 H160S

160 H160T 1.01 0.91 160 H160V 0.76 0.74 160 H160W 0.26 0.66 160 H160Y 0.86 0.89 165 D165A 0.53 0.12 165 D165C 1.01 0.07 165 D165E 1.14 0.07 165 D165F 0.09 0.07 165 D165G 0.63 0.20 165 D165H 0.46 0.18 165 D165I 0.06 0.15 165 D165K 0.07 0.14 165 D165L 0.30 0.11 165 D165M 0.58 0.10 165 D165N 1.16 1.10 165 D165P

165 D165Q 0.53 0.11 165 D165R 0.08 0.11 165 D165S 0.83 0.43 165 D165T

0.50 165 D165V

0.15 165 D165W

165 D165Y 0.31 0.07 168 E168A 0.83 0.92 168 E168C 0.83 0.50 168 E168D 0.82 0.57 168 E168F 0.69 0.59 168 E168G 0.92 0.75 168 E168H 0.84 0.90 168 E168I 1.08 0.71 168 E168K

168 E168L 0.80 0.92 168 E168M 1.12 0.80 168 E168N 0.97 0.83 168 E168P

168 E168Q 0.88 0.87 168 E168R 1.18 0.90 168 E168S 0.95 0.83 168 E168T 0.83 0.16 168 E168V 0.89 0.73 168 E168W 1.23 0.66 168 E168Y 0.76 0.82 172 L172A 1.14 1.06 172 L172C 1.07 0.89 172 L172D 0.83 0.91 172 L172E 0.97 1.01 172 L172G 0.50 0.60 172 L172H 0.93 1.06 172 L172I 0.97 0.90 172 L172K 0.98 1.12 172 L172M 0.86 0.91 172 L172N 0.91 0.96 172 L172P 0.17 0.83 172 L172Q 1.00 0.89 172 L172R 1.16 1.06 172 L172S 0.78 1.01 172 L172T 0.82 0.94 172 L172V 1.02 0.88 172 L172W 1.09 0.92 172 L172Y 1.06 0.98 173 S173A 0.92 0.74 173 S173C 0.82 0.57 173 S173D 0.63 0.71 173 S173E 1.07 0.65 173 S173F 0.82 0.25 173 S173G 0.73 0.78 173 S173H 0.85 0.66 173 S173I 1.20 0.59 173 S173K 1.17 1.03 173 S173L 0.75 0.20 173 S173M 1.05 0.48 173 S173N 1.02 0.84 173 S173Q 1.08 0.84 173 S173R 0.88 1.03 173 S173T 1.33 0.86 173 S173V 1.12 0.46 173 S173W 0.86 0.20 173 S173Y 0.90 0.25 177 K177A

1.20 177 K177C

0.76 177 K177D

1.07 177 K177E

1.08 177 K177F

1.01 177 K177G

1.03 177 K177H

1.07 177 K177I

0.89 177 K177L 0.89 0.91 177 K177M 0.10 0.90 177 K177N

1.15 177 K177P

1.11 177 K177Q 0.08 1.07 177 K177R 0.47 1.09 177 K177S

1.00 177 K177T

1.01 177 K177W

1.07 177 K177Y

0.97 188 E188A

1.10 188 E188C

0.85 188 E188D

1.21 188 E188F

1.08 188 E188G

1.17 188 E188H

1.00 188 E188I

1.11 188 E188K

1.02 188 E188M

1.08 188 E188N

1.06 188 E188P 1.40 1.16 188 E188Q

1.06 188 E188S

1.10 188 E188T

1.17 188 E188V

1.08 188 E188W

1.07 188 E188Y

1.02 191 T191A 0.49 1.11 191 T191C 0.13 1.07 191 T191D 0.91 1.03 191 T191F

1.02 191 T191G 0.19 1.09 191 T191H

1.27 191 T191I 0.18 1.06 191 T191K

1.33 191 T191L

1.08 191 T191M 0.06 1.09 191 T191N 0.76 1.13 191 T191P 0.99 1.07 191 T191Q 0.18 1.17 191 T191R

1.20 191 T191S 0.72 1.05 191 T191V 0.16 1.02 191 T191W

0.91 192 E192A

1.26 192 E192C 0.55 1.12 192 E192D 0.42 1.50 192 E192G

1.38 192 E192H

0.78 192 E192I

1.00 192 E192K

0.33 192 E192M

1.19 192 E192N

1.35 192 E192P

1.33 192 E192Q 0.22 1.55 192 E192R

0.37 192 E192S

1.47 192 E192T 0.10 1.35 192 E192V

1.25 192 E192W

1.17 192 E192Y

1.30 193 N193A

0.98 193 N193C 0.73 0.62 193 N193D

0.95 193 N193E

0.74 193 N193F 1.71 0.98 193 N193G

0.96 193 N193H 1.10 0.92 193 N193I

0.78 193 N193K 1.28 1.17 193 N193L 1.22 0.78 193 N193M 0.81 0.96 193 N193P

0.90 193 N193R 0.87 0.97 193 N193S

1.15 193 N193T

0.86 193 N193W 1.09 0.73 193 N193Y 1.89 1.10 196 Y196A 0.74 1.57 196 Y196C

1.36 196 Y196D 0.29 1.29 196 Y196E

1.29 196 Y196F 0.74 1.38 196 Y196G

1.09 196 Y196H

1.92 196 Y196I

1.61 196 Y196K

1.29 196 Y196L

1.14 196 Y196N 0.54 0.94 196 Y196P

1.50 196 Y196R

1.29 196 Y196S 0.36 1.59 196 Y196T

1.65 196 Y196V

1.55 196 Y196W

0.57 199 L199A 0.16 0.42 199 L199E

0.34 199 L199G

0.31 199 L199H

0.18 199 L199I 0.14 0.30 199 L199K 0.22 0.15 199 L199M 0.30 0.14 199 L199N

0.07 199 L199P

199 L199Q

0.20 199 L199R

0.23 199 L199S

0.29 199 L199T 0.12 0.35 199 L199V 0.61 0.13 199 L199W

199 L199Y

200 M200A 1.03 0.68 200 M200C 0.84 0.53 200 M200D 0.71 0.81 200 M200E 0.54 0.55 200 M200F 0.05 0.25 200 M200G 0.23 0.41 200 M200H

200 M200I 1.14 0.57 200 M200K

200 M200L 0.68 1.11 200 M200N 0.46 0.72 200 M200P

200 M200Q 0.78 0.77 200 M200S 0.61 1.11 200 M200T 0.80 0.61 200 M200V 0.97 0.56 200 M200W

201 Y201A 0.90 1.41 201 Y201C 1.22 0.14 201 Y201D 0.60 0.73 201 Y201E 0.81 1.36 201 Y201F 0.85 0.81 201 Y201G 0.56 1.63 201 Y201H 1.06 1.44 201 Y201I 1.35 0.11 201 Y201K 0.89 0.08 201 Y201L 1.05 0.18 201 Y201M 1.16 1.21 201 Y201N 1.15 0.31 201 Y201P

201 Y201Q 1.11 0.79 201 Y201R 0.87 0.06 201 Y201S 0.74 1.11 201 Y201T 0.65 0.39 201 Y201V

201 Y201W 0.73 0.08 202 A202C 0.97 0.57 202 A202D 0.83 0.93 202 A202E 0.49 0.85 202 A202F

0.68 202 A202G 0.45 0.83 202 A202H

1.30 202 A202I 0.50 1.02 202 A202K 0.37 0.12 202 A202L 0.46 0.95 202 A202M 0.32 0.84 202 A202N 0.53 1.08 202 A202P

0.72 202 A202Q 0.47 1.01 202 A202R

202 A202S 0.69 0.79 202 A202T 0.63 1.07 202 A202V 0.82 1.02 202 A202Y

0.43 213 T213A 1.11 0.98 213 T213C 0.97 0.77 213 T213D 1.12 0.91 213 T213E 1.11 0.88 213 T213F 1.13 0.75 213 T213G 1.11 0.91 213 T213H 0.92 1.00 213 T213I

213 T213K 0.90 1.11 213 T213L 1.26 0.75 213 T213M 1.26 0.78 213 T213N 1.11 0.91 213 T213P 0.94 0.91 213 T213Q 1.12 1.02 213 T213R 1.05 1.05 213 T213S 1.10 1.08 213 T213V 1.35 0.76 213 T213W 1.17 0.68 216 K216A 0.66 0.24 216 K216D

216 K216E 1.03 1.30 216 K216F

216 K216G 0.83 1.20 216 K216H 0.90 1.28 216 K216I

216 K216L

216 K216M 0.97 1.39 216 K216P 0.91 0.97 216 K216Q 1.04 1.34 216 K216R 0.77 1.32 216 K216S 0.97 1.28 216 K216T 0.99 1.22 216 K216V 0.95 1.07 216 K216W 1.00 1.13 216 K216Y 0.79 1.31 217 N217A 1.10 0.87 217 N217C 0.81 0.78 217 N217E

0.73 217 N217F 0.90 0.88 217 N217G 0.95 0.90 217 N217H 1.09 0.90 217 N217I 1.08 0.76 217 N217L 1.09 0.82 217 N217M 0.97 0.80 217 N217P 0.97 0.73 217 N217Q 1.31 0.74 217 N217R 1.19 0.87 217 N217S 1.05 0.87 217 N217T 1.01 0.87 217 N217V 1.18 0.69 217 N217W 0.99 0.80 217 N217Y

220 K220A 1.06 0.79 220 K220C 1.05 0.75 220 K220D 1.02 0.88 220 K220E 1.12 0.88 220 K220F 1.03 0.78 220 K220G 1.10 0.84 220 K220H 1.12 0.81 220 K220I 1.13 0.81 220 K220M 1.05 0.75 220 K220N 1.17 0.80 220 K220P 1.33 0.89 220 K220Q 1.21 0.87 220 K220R 1.26 0.83 220 K220S 1.30 0.81 220 K220T

0.09 220 K220V 1.21 0.82 220 K220W 1.01 0.81 220 K220Y 1.08 0.84 221 W221A 0.88 1.54 221 W221C 0.95 1.09 221 W221D 0.84 1.31 221 W221E

221 W221F 1.07 1.28 221 W221G

221 W221H

221 W221I 1.33 1.23 221 W221K

221 W221L 0.88 1.50 221 W221M 1.16 1.35 221 W221N 1.11 1.57 221 W221P

221 W221R 0.93 1.29 221 W221S 1.34 1.40 221 W221V 1.13 1.31 221 W221Y 1.14 1.36 227 N227A 1.01 1.02 227 N227C 0.92 0.95 227 N227D 1.01 1.06 227 N227E 1.03 1.06 227 N227F 0.72 0.81 227 N227G 1.05 1.09 227 N227H 0.95 1.13 227 N227I 1.03 0.76 227 N227K 1.00 1.13 227 N227L 0.84 0.75 227 N227M 0.84 0.87 227 N227P 1.08 0.88 227 N227Q 0.94 1.00 227 N227R 0.89 1.03 227 N227S 0.96 0.95 227 N227T 1.06 0.96 227 N227V 1.05 0.84 227 N227W 1.07 0.81 227 N227Y 1.01 0.85 232 R232A

232 R232C 0.40 0.14 232 R232D

232 R232E 0.41 0.12 232 R232G 0.06 0.23 232 R232H 0.66 0.34 232 R232K 0.52 0.47 232 R232M 0.62 0.12 232 R232N

232 R232P

232 R232Q 0.54 0.12 232 R232S 0.59 0.16 232 R232T 0.76 0.17 232 R232V 0.70 0.15 232 R232W

232 R232Y

235 A235C 0.86 0.53 235 A235D 0.70 0.98 235 A235E 0.93 0.84 235 A235F 1.01 0.68 235 A235G 1.17 0.78 235 A235H 0.80 1.01 235 A235I 1.07 0.84 235 A235K 0.93 1.14 235 A235L 0.89 0.97 235 A235M 0.99 0.91 235 A235N 0.78 1.03 235 A235P 0.97 0.48 235 A235Q 1.01 0.89 235 A235R 1.03 1.14 235 A235S 0.92 1.00 235 A235T

235 A235V 1.01 0.86 235 A235W 0.98 0.60 235 A235Y 0.91 0.93 237 K237A

0.78 237 K237C

0.57 237 K237D

0.08 237 K237E

0.74 237 K237F

0.09 237 K237G

1.21 237 K237H

0.26 237 K237I

0.40 237 K237L

0.57 237 K237M

0.46 237 K237N

0.43 237 K237P

0.30 237 K237Q

0.77 237 K237R 0.48 0.88 237 K237T

0.69 237 K237V

0.54 237 K237W

237 K237Y

238 H238A

0.62 238 H238C

0.63 238 H238D

0.75 238 H238F

238 H238G

0.73 238 H238I

0.18 238 H238K

238 H238L

0.25 238 H238M

0.36 238 H238N 0.21 0.83 238 H238P

0.57 238 H238Q

1.18 238 H238R

238 H238T

0.74 238 H238V

0.52 238 H238Y

240 K240A

1.13 240 K240D

1.19 240 K240E

1.19 240 K240F

0.90 240 K240G

1.22 240 K240H

1.17 240 K240I

0.99 240 K240M 0.31 1.13 240 K240N

1.37 240 K240P

1.69 240 K240Q 0.12 1.21 240 K240R 0.27 1.41 240 K240S

1.07 240 K240T

1.23 240 K240V

1.09 240 K240W

1.01 240 K240Y

1.11 246 D246A 0.73 1.03 246 D246E 1.18 1.03 246 D246F 0.67 1.02 246 D246G 0.61 1.09 246 D246H 0.71 1.05 246 D246I 0.75 0.85 246 D246K 0.36 1.18 246 D246L 0.81 0.91 246 D246M 0.80 0.92 246 D246N 0.68 0.97 246 D246P 0.47 0.81 246 D246Q 0.78 0.98 246 D246R 0.24 1.31 246 D246S 0.97 1.01 246 D246T 0.83 1.14 246 D246Y 0.90 0.96 249 S249A 1.06 0.97 249 S249C 0.93 0.74 249 S249D 0.98 0.94 249 S249E 1.27 0.92 249 S249F 0.91 0.74 249 S249G 0.91 0.94 249 S249H 1.04 0.93 249 S249K 1.15 1.02 249 S249L 1.14 0.82 249 S249M 0.95 0.77 249 S249P 1.09 0.80 249 S249Q 1.20 0.94 249 S249R 1.07 1.03 249 S249T 1.17 0.91 249 S249V 1.01 0.74 249 S249W 1.13 0.77 249 S249Y 1.07 0.87 250 Y250A 0.99 1.21 250 Y250C 1.03 1.12 250 Y250D 0.97 1.29 250 Y250E 1.13 1.33 250 Y250F 1.29 1.28 250 Y250G 1.09 1.33 250 Y250I 1.35 1.27 250 Y250K 1.07 1.48 250 Y250L 1.02 1.32 250 Y250M 1.35 1.39 250 Y250N 1.05 1.40 250 Y250P 0.71 1.05 250 Y250Q 1.01 1.54 250 Y250R 0.99 1.55 250 Y250S 1.02 1.41 250 Y250T

250 Y250V

250 Y250W 0.99 1.35 252 R252A 1.12 1.08 252 R252C 0.97 0.81 252 R252D 0.89 0.86 252 R252E 1.09 1.12 252 R252F 1.01 0.89 252 R252G 0.76 1.00 252 R252I 1.07 0.97 252 R252K 1.19 1.21 252 R252L 1.32 0.96 252 R252M 0.98 0.96 252 R252N 1.15 0.97 252 R252P 0.72 0.83 252 R252Q 1.16 1.04 252 R252S 1.04 1.01 252 R252T 1.09 0.99 252 R252V 1.01 0.94 252 R252Y 1.14 0.86 253 S253A 1.09 0.97 253 S253D 1.07 1.04 253 S253E

253 S253F 1.19 0.82 253 S253G 1.18 0.92 253 S253H 1.13 0.97 253 S253I 1.13 0.84 253 S253K 1.10 1.01 253 S253L 1.09 0.79 253 S253M

253 S253N 1.06 1.03 253 S253P 0.95 0.90 253 S253Q 1.13 0.93 253 S253T 1.14 0.97 253 S253V 1.15 0.90 253 S253W 1.04 0.87 253 S253Y 1.34 0.94 254 Q254A 0.98 0.88 254 Q254C 0.94 0.66 254 Q254D 1.10 0.90 254 Q254E 1.29 0.89 254 Q254F 1.23 0.74 254 Q254G 1.15 0.77 254 Q254H 1.04 0.94 254 Q254I 1.12 0.91 254 Q254K 1.00 0.99 254 Q254L 1.09 0.82 254 Q254M 0.94 0.89 254 Q254N 1.17 0.90 254 Q254R 1.05 0.98 254 Q254S 1.07 0.98 254 Q254T 1.21 0.65 254 Q254V 1.31 0.92 254 Q254W 1.17 0.69 254 Q254Y 1.03 0.87 255 T255A 1.09 0.73 255 T255C 0.89 0.78 255 T255D

255 T255E 1.09 0.64 255 T255F 1.30 0.68 255 T255G 1.15 0.73 255 T255H 1.10 0.74 255 T255I 1.18 0.70 255 T255K 1.27 0.83 255 T255L 0.97 0.73 255 T255M 0.98 0.72 255 T255N 0.83 0.76 255 T255P 0.77 0.59 255 T255R 1.12 0.85 255 T255S 1.10 0.84 255 T255V 1.17 0.70 255 T255W 1.27 0.74 255 T255Y 1.02 0.72 257 K257A 1.08 0.67 257 K257C 0.89 0.49 257 K257D 1.16 0.75 257 K257E 1.15 0.76 257 K257F 1.03 0.92 257 K257G 0.97 0.73 257 K257H 1.12 0.69 257 K257I 1.09 0.59 257 K257L 1.26 0.74 257 K257M 1.29 0.79 257 K257N 1.16 0.83 257 K257P 0.62 0.38 257 K257Q 1.18 0.82 257 K257R 1.03 0.89 257 K257S 1.29 0.71 257 K257T 1.04 0.77 257 K257V 1.31 0.78 257 K257W 0.99 0.72 258 P258A 0.97 1.08 258 P258C 1.17 0.85 258 P258D 1.33 1.10 258 P258E 0.95 1.05 258 P258F 0.96 0.75 258 P258G 1.30 1.02 258 P258H 1.38 1.13 258 P258I 1.27 0.25 258 P258K 1.29 1.11 258 P258L 1.08 0.61 258 P258M 1.09 0.91 258 P258N 1.07 1.01 258 P258Q 1.31 1.13 258 P258R 1.02 1.13 258 P258S 1.12 1.08 258 P258T 1.27 1.10 258 P258V 1.29 0.80 258 P258W 1.14 0.87 258 P258Y 1.16 1.08 268 Y268A 0.86 1.39 268 Y268C 0.47 1.10 268 Y268D 0.59 1.44 268 Y268E 0.55 1.47 268 Y268F 1.28 1.07 268 Y268G 1.03 1.21 268 Y268H 0.87 1.24 268 Y268I

268 Y268K 0.78 1.90 268 Y268L 0.72 1.10 268 Y268M 0.97 1.15 268 Y268N 0.69 1.51 268 Y268P 0.78 1.41 268 Y268Q 0.71 1.30 268 Y268R 0.76 1.49 268 Y268S 1.06 1.22 268 Y268T 0.99 1.12 268 Y268V 0.88 0.99 268 Y268W 0.97 1.07 272 K272A

0.73 272 K272C

0.68 272 K272D

0.86 272 K272E

0.85 272 K272F

0.56 272 K272G

0.60 272 K272H

0.78 272 K272I

0.81 272 K272M

0.77 272 K272N

0.67 272 K272P

0.38 272 K272R 0.90 0.86 272 K272S

0.79 272 K272T

0.99 272 K272V

0.64 272 K272W

0.48 272 K272Y

0.66 274 H274A 0.66 1.40 274 H274C 0.65 0.68 274 H274D 0.64 1.20 274 H274E 0.86 1.14 274 H274F 0.88 1.00 274 H274G 0.56 1.36 274 H274I 0.76 1.39 274 H274K 0.85 1.60 274 H274L 0.87 1.40 274 H274N 0.67 1.50 274 H274P

0.50 274 H274Q 0.84 1.47 274 H274R 0.80 1.50 274 H274S 0.67 1.28 274 H274T 0.69 1.38 274 H274W 1.26 0.79 274 H274Y 1.05 1.07 275 N275A 0.32 1.01 275 N275C 0.22 0.68 275 N275D 0.08 1.03 275 N275E

0.98 275 N275F

0.81 275 N275G 0.18 1.00 275 N275H 0.60 1.10 275 N275I 0.15 0.87 275 N275K 0.22 1.22 275 N275L 0.20 1.02 275 N275M 0.20 0.96 275 N275P

0.37 275 N275Q 0.21 0.96 275 N275R 0.28 1.04 275 N275S 0.28 0.92 275 N275T 0.19 0.79 275 N275V 0.19 0.76 275 N275W 0.05 0.76 275 N275Y 0.89 0.93 279 K279C

0.54 279 K279E

0.91 279 K279F

0.70 279 K279G

0.85 279 K279H

0.36 279 K279I

0.68 279 K279L

0.52 279 K279M

0.71 279 K279N

0.91 279 K279P

0.46 279 K279Q

0.86 279 K279S

0.94 279 K279T

0.92 279 K279V

0.78 279 K279W

0.76 279 K279Y

0.78 283 T283A 1.06 0.97 283 T283C 1.16 0.78 283 T283D 0.92 1.03 283 T283E 0.95 1.01 283 T283G 0.97 1.01 283 T283H 1.09 0.84 283 T283I 1.10 0.72 283 T283K 1.14 1.01 283 T283L 1.07 0.76 283 T283M 1.26 0.93 283 T283N 1.29 0.96 283 T283P 0.46 0.56 283 T283R 0.82 1.08 283 T283S 1.02 1.06 283 T283V 1.23 0.81 283 T283W 1.07 0.75 283 T283Y 1.01 1.04 285 S285A 0.93 0.80 285 S285C 0.73 0.61 285 S285D 0.91 1.09 285 S285E 1.33 0.89 285 S285F 1.18 1.02 285 S285H 0.98 1.10 285 S285I 0.84 0.52 285 S285K 1.16 0.84 285 S285L 0.85 0.54 285 S285M 0.98 0.76 285 S285Q 1.38 1.22 285 S285R 0.84 0.96 285 S285T 0.98 0.79 285 S285V 0.70 0.63 285 S285W 1.13 1.08 285 S285Y 0.97 1.49 293 N293A 1.02 0.93 293 N293C 0.78 0.69 293 N293D 1.08 0.89 293 N293E 0.87 0.92 293 N293F 0.89 0.70 293 N293G 1.31 0.92 293 N293H 1.12 1.05 293 N293I 0.94 0.75 293 N293K 1.42 1.41 293 N293L 0.87 0.81 293 N293M 0.95 1.07 293 N293P 0.97 0.40 293 N293Q 1.14 1.06 293 N293R 0.86 1.37 293 N293S 0.93 0.95 293 N293T 1.10 1.12 293 N293V 1.04 0.82 293 N293W 1.09 0.78 293 N293Y 1.19 0.74 294 K294A 0.83 0.92 294 K294C 0.83 0.50 294 K294D 0.82 0.57 294 K294E 0.83 0.68 294 K294F 0.69 0.59 294 K294G 0.92 0.75 294 K294H 0.84 0.90 294 K294I 1.08 0.71 294 K294L 0.80 0.92 294 K294M 1.12 0.80 294 K294N 0.97 0.83 294 K294P

294 K294Q 0.88 0.87 294 K294R 1.18 0.90 294 K294S 0.95 0.83 294 K294T 0.83 0.16 294 K294V 0.89 0.73 294 K294W 1.23 0.66 294 K294Y 0.76 0.82 297 T297C 0.86 0.53 297 T297D 0.70 0.98 297 T297E 0.93 0.84 297 T297F 1.01 0.68 297 T297G 1.17 0.78 297 T297H 0.80 1.01 297 T297I 1.07 0.84 297 T297K 0.93 1.14 297 T297L 0.89 0.97 297 T297M 0.99 0.91 297 T297N 0.78 1.03 297 T297P 0.97 0.48 297 T297Q 1.01 0.89 297 T297R 1.03 1.14 297 T297S 0.92 1.00 297 T297V 1.01 0.86 297 T297W 0.98 0.60 297 T297Y 0.91 0.93 300 K300A 0.99 0.79 300 K300C 0.95 0.39 300 K300D 0.91 0.61 300 K300E 0.86 0.78 300 K300F 0.74 0.63 300 K300G 0.98 0.62 300 K300H 1.04 0.83 300 K300I 1.02 0.82 300 K300L 0.91 0.73 300 K300M 1.17 0.80 300 K300N 1.02 0.80 300 K300P

300 K300Q 0.90 0.86 300 K300R 1.20 0.92 300 K300S 0.93 0.80 300 K300T 1.16 0.87 300 K300V 1.15 0.84 300 K300W 0.97 0.57 301 S301A 1.10 0.89 301 S301E 1.12 0.94 301 S301F 1.44 0.68 301 S301G 1.02 1.05 301 S301H 1.12 0.87 301 S301I 1.28 0.74 301 S301K 1.08 1.05 301 S301L 1.09 0.97 301 S301M 1.09 0.87 301 S301N 1.16 0.64 301 S301P 1.21 0.61 301 S301Q 1.18 0.95 301 S301R 1.35 0.89 301 S301T 1.23 0.85 301 S301V 1.18 0.81 301 S301W 1.27 0.75 301 S301Y 1.10 0.80 306 D306A 0.82 0.40 306 D306C 0.74 0.30 306 D306E 0.80 0.71 306 D306F 0.71 0.10 306 D306G 0.76 0.26 306 D306H 0.84 0.35 306 D306I 0.80 0.18 306 D306K 0.77 0.41 306 D306L 0.78 0.18 306 D306N 1.15 0.89 306 D306P 0.82 0.39 306 D306Q 1.03 0.43 306 D306R 0.82 0.27 306 D306S 0.81 0.50 306 D306T 0.88 0.29 306 D306V 0.99 0.22 306 D306W

306 D306Y 0.94 0.12 309 T309A

309 T309C 1.15 0.59 309 T309D 1.27 0.89 309 T309E 0.95 0.91 309 T309F 1.15 0.80 309 T309G 1.17 1.00 309 T309H 0.94 0.97 309 T309I 1.17 0.82 309 T309K 1.18 1.08 309 T309L 1.15 0.95 309 T309M 1.15 0.97 309 T309N 1.20 0.99 309 T309P 0.93 0.20 309 T309Q 1.19 0.98 309 T309R 1.12 1.08 309 T309S 1.00 1.04 309 T309V 1.38 0.95 309 T309W 1.08 0.77 309 T309Y 1.11 0.94 312 T312A 1.01 1.00 312 T312C 0.99 0.70 312 T312D 1.03 0.96 312 T312E 1.15 0.95 312 T312F 1.05 0.92 312 T312G 1.18 1.07 312 T312H 1.30 0.99 312 T312K 0.83 0.25 312 T312L 1.08 0.95 312 T312M 0.98 0.91 312 T312N 1.04 0.99 312 T312P 0.74 0.85 312 T312Q 1.05 0.94 312 T312R 1.13 1.00 312 T312S 1.29 0.99 312 T312V 1.40 0.87 312 T312W 1.14 0.83 312 T312Y 1.31 0.92 313 N313A 1.01 0.93 313 N313C 0.95 0.63 313 N313D 0.95 0.51 313 N313E 1.05 0.90 313 N313F 1.06 0.64 313 N313G 1.25 0.96 313 N313H 1.25 0.94 313 N313I 1.44 0.55 313 N313K 1.12 0.85 313 N313L 1.21 0.85 313 N313M 1.02 0.89 313 N313P 1.05 0.81 313 N313Q 1.00 1.00 313 N313R 1.19 1.13 313 N313S 1.25 1.05 313 N313V 1.28 0.74 313 N313W 1.01 0.67 313 N313Y 1.10 0.90 317 K317A 0.98 0.94 317 K317C 0.83 0.54 317 K317D 0.82 0.86 317 K317E 0.78 0.91 317 K317F 0.92 0.84 317 K317G 0.91 0.88 317 K317L 1.10 0.86 317 K317M 1.02 0.95 317 K317N 1.03 0.92 317 K317P 0.86 0.80 317 K317Q 0.76 0.94 317 K317R 0.78 0.89 317 K317S 1.04 0.93 317 K317T 0.94 0.88 317 K317V 1.00 0.93 317 K317W 1.08 0.83 317 K317Y 1.05 0.93 318 D318A 0.93 1.38 318 D318E 0.90 1.09 318 D318F 0.78 1.22 318 D318G 0.91 1.39 318 D318H 1.12 1.10 318 D318I 0.75 1.40 318 D318K 0.65 1.73 318 D318L 1.00 1.31 318 D318M 0.90 1.26 318 D318N 0.92 1.19 318 D318P 0.61 0.37 318 D318Q 0.93 1.14 318 D318R 0.71 1.54 318 D318S 1.11 1.37 318 D318T 1.40 1.32 318 D318V 0.81 1.34 318 D318W 0.90 1.07 318 D318Y 1.10 1.33 319 Q319A 1.02 1.13 319 Q319C 0.73 1.38 319 Q319D 0.85 1.31 319 Q319E 0.98 1.20 319 Q319F 0.87 1.11 319 Q319G 1.14 1.03 319 Q319H 0.94 1.28 319 Q319I 0.94 1.32 319 Q319K 1.10 1.52 319 Q319L 0.95 1.11 319 Q319M 0.90 1.09 319 Q319N 0.91 1.12 319 Q319P 1.13 0.57 319 Q319R 1.18 1.44 319 Q319S 0.91 1.12 319 Q319T 0.98 1.10 319 Q319V 1.07 1.08 319 Q319W 1.05 1.08 319 Q319Y 1.04 1.41 320 P320A 1.02 0.96 320 P320C 1.01 0.75 320 P320D 0.74 0.91 320 P320E 1.04 0.85 320 P320F 0.76 0.77 320 P320G 1.00 1.00 320 P320H 1.00 1.18 320 P320I 0.86 0.80 320 P320K 0.96 1.23 320 P320L 0.87 0.83 320 P320M 1.04 0.60 320 P320Q 0.95 1.08 320 P320R 0.79 1.25 320 P320S 1.16 1.03 320 P320T 1.11 1.28 320 P320V 1.08 0.88 320 P320W 0.90 1.03 320 P320Y 1.05 1.03 338 L338A 1.36 1.29 338 L338C 1.24 0.67 338 L338D 1.00 0.94 338 L338E 0.87 0.65 338 L338F 0.90 0.17 338 L338G 1.38 1.34 338 L338H

338 L338I 1.12 1.32 338 L338K

338 L338M 1.20 1.27 338 L338P 1.11 1.23 338 L338Q 0.96 0.61 338 L338R

338 L338S 1.13 1.51 338 L338T 1.42 1.05 338 L338V 1.14 1.55 338 L338W 0.98 0.14 338 L338Y 1.15 0.11 339 Q339A 1.08 1.13 339 Q339C 0.88 0.79 339 Q339D 0.93 0.11 339 Q339E 1.07 0.84 339 Q339F 0.86 0.55 339 Q339G 1.17 1.21 339 Q339H 1.03 1.04 339 Q339K 1.26 1.13 339 Q339L 1.12 0.70 339 Q339M 0.93 0.81 339 Q339P 1.02 1.24 339 Q339R 0.81 0.35 339 Q339S 1.02 1.02 339 Q339T 1.35 1.01 339 Q339V 1.23 0.76 339 Q339W

339 Q339Y 1.14 0.78 340 S340A 1.23 1.43 340 S340C 0.74 0.75 340 S340D 0.97 1.63 340 S340E 0.92 1.58 340 S340F 0.83 0.82 340 S340H 1.12 1.45 340 S340I 1.07 1.07 340 S340K 0.99 1.76 340 S340L

340 S340M 1.24 1.20 340 S340N 1.10 1.75 340 S340P 0.69 0.81 340 S340Q 1.21 1.76 340 S340T 1.21 1.14 340 S340V 1.00 1.09 340 S340Y 1.02 0.97 343 D343A 0.96 0.35 343 D343C 1.32 0.74 343 D343E 1.00 1.07 343 D343F 0.91 0.79 343 D343H 0.98 1.02 343 D343I 1.27 0.88 343 D343L 0.95 1.08 343 D343M 0.99 1.02 343 D343N 1.05 0.88 343 D343P 1.30 1.03 343 D343Q 1.14 1.01 343 D343R 1.25 1.03 343 D343T 1.08 0.98 343 D343W 1.00 0.64 343 D343Y 1.29 0.82 345 W345A 1.05 0.90 345 W345C 0.97 0.43 345 W345D 1.10 1.15 345 W345E 1.06 1.24 345 W345F 1.07 0.55 345 W345H 1.15 1.10 345 W345I 1.28 0.90 345 W345K

345 W345L 1.07 0.99 345 W345M 1.02 1.01 345 W345N 1.07 1.10 345 W345P 1.00 0.94 345 W345Q 1.26 1.10 345 W345S 1.01 1.12 345 W345T 1.15 1.15 345 W345V 1.16 1.02 363 C363A 0.84 1.06 363 C363D 0.87 1.74 363 C363E 0.99 1.34 363 C363F 0.83 1.03 363 C363G 0.61 0.83 363 C363H 0.78 0.76 363 C363I 0.92 0.63 363 C363L 0.73 0.89 363 C363M 0.97 1.36 363 C363N 0.92 1.86 363 C363P

0.05 363 C363Q 0.88 1.78 363 C363R

363 C363S 0.88 1.35 363 C363T 1.15 0.18 363 C363V 1.02 0.99 363 C363W 0.35 0.70 363 C363Y 0.92 0.12 366 Y366A 0.96 1.14 366 Y366C 0.46 0.37 366 Y366D 0.52 1.18 366 Y366E 0.91 1.18 366 Y366F 0.91 0.87 366 Y366G 0.94 1.08 366 Y366H 1.07 1.12 366 Y366I 0.85 0.87 366 Y366K 0.72 0.82 366 Y366L 0.77 0.61 366 Y366M 0.92 0.79 366 Y366N 1.03 0.91 366 Y366P 0.54 0.78 366 Y366Q 1.03 1.49 366 Y366R 0.96 0.96 366 Y366S 1.07 1.02 366 Y366T 1.01 0.91 366 Y366V 1.04 0.94 366 Y366W 1.11 0.99 369 Y369A

369 Y369C 0.44 0.16 369 Y369E 0.98 0.87 369 Y369F 1.03 0.79 369 Y369G 0.86 0.33 369 Y369H 0.89 0.78 369 Y369I 1.33 0.91 369 Y369K 1.07 0.80 369 Y369M 1.06 1.02 369 Y369P 0.49 0.20 369 Y369Q 1.07 0.79 369 Y369R 1.11 0.95 369 Y369S 0.89 0.60 369 Y369T 1.28 0.68 369 Y369V 1.17 0.91 369 Y369W 1.09 0.95 370 Y370A 1.03 1.21 370 Y370C 0.44 0.19 370 Y370D 0.48 1.35 370 Y370E 0.98 1.35 370 Y370F 0.90 0.73 370 Y370G 1.21 1.18 370 Y370H 0.96 1.36 370 Y370I 0.99 1.00 370 Y370K 0.93 1.65 370 Y370L 0.93 0.88 370 Y370M 0.91 1.04 370 Y370N 1.04 1.41 370 Y370P 0.44 0.67 370 Y370Q 0.87 1.51 370 Y370S 1.06 1.50 370 Y370T 1.07 1.10 370 Y370V 1.05 1.13 370 Y370W 0.94 0.91 375 Y375A 1.03 1.39 375 Y375C 0.59 0.48 375 Y375D 0.96 1.52 375 Y375E 0.96 1.48 375 Y375F 0.90 1.00 375 Y375G 0.90 0.98 375 Y375H 0.98 1.16 375 Y375I 0.94 1.06 375 Y375K 0.96 1.43 375 Y375L 1.03 1.07 375 Y375M 0.98 1.05 375 Y375N 0.92 1.48 375 Y375P 0.92 0.89 375 Y375Q 0.92 1.56 375 Y375R 0.77 1.61 375 Y375S 0.92 1.29 375 Y375T 1.25 1.04 375 Y375W 0.98 0.88 379 S379A 1.01 1.02 379 S379C 0.60 0.44 379 S379D 0.92 0.96 379 S379E 0.99 1.01 379 S379F 0.48 0.43 379 S379G 0.90 0.91 379 S379H

379 S379I 0.80 0.70 379 S379K 1.00 1.12 379 S379L 0.84 0.56 379 S379M 0.87 0.80 379 S379N 1.03 0.98 379 S379P 0.61 0.39 379 S379Q 0.94 0.98 379 S379R 0.96 1.01 379 S379T 1.07 0.95 379 S379V 0.90 0.75 379 S379W 0.70 0.35 379 S379Y 0.92 0.59 381 K381A 0.85 0.78 381 K381C 0.86 0.35 381 K381D 0.87 0.65 381 K381E 0.93 0.81 381 K381F 0.96 0.20 381 K381G 0.96 0.82 381 K381H 1.13 0.73 381 K381I 0.98 0.36 381 K381L 0.95 0.38 381 K381M 0.93 0.56 381 K381N 0.87 0.68 381 K381P 1.18 0.39 381 K381Q 1.03 0.90 381 K381R 1.20 0.95 381 K381S 1.18 0.89 381 K381T 1.01 0.60 381 K381V 1.00 0.43 381 K381W 0.90 0.22 381 K381Y 0.87 0.63 385 D385A 1.01 0.88 385 D385C

385 D385E 0.89 1.05 385 D385F 0.73 0.54 385 D385G 1.05 0.88 385 D385H 0.96 0.99 385 D385I 0.46 0.15 385 D385K 1.00 1.06 385 D385L 0.96 0.47 385 D385N 0.91 0.96 385 D385P

385 D385Q 1.02 1.01 385 D385R 0.86 0.95 385 D385S 1.10 1.00 385 D385T 1.22 0.92 385 D385V 0.85 0.43 385 D385W 0.98 0.53 386 P386A 0.90 0.80 386 P386C 0.72 0.69 386 P386D 0.85 0.94 386 P386E 0.94 0.87 386 P386F 0.72 0.66 386 P386G 1.02 0.77 386 P386H 0.89 0.93 386 P386I 1.12 0.73 386 P386K 1.22 0.87 386 P386L 0.96 0.73 386 P386M 0.94 0.70 386 P386N 0.91 0.86 386 P386Q 0.95 0.86 386 P386S 0.83 0.82 386 P386T 1.00 0.54 386 P386V 1.11 0.79 386 P386W 0.90 0.44 386 P386Y 0.91 0.78 391 R391A 0.58 0.22 391 R391C 0.28 0.12 391 R391E 0.05 0.08 391 R391G 0.42 0.16 391 R391H 0.59 0.29 391 R391K 0.88 0.59 391 R391L

391 R391N 0.71 0.38 391 R391P

391 R391Q 0.62 0.28 391 R391S

0.33 391 R391T 0.67 0.25 391 R391V 0.24 0.09 391 R391W

391 R391Y

392 R392A 0.89 0.73 392 R392C 0.74 0.66 392 R392E 0.79 0.46 392 R392F 1.03 0.43 392 R392G 0.99 0.65 392 R392H 0.86 0.96 392 R392I 1.08 0.57 392 R392K 1.10 1.09 392 R392L 0.91 0.63 392 R392M 1.07 0.72 392 R392N 0.89 0.90 392 R392P 0.67 0.31 392 R392Q 1.12 0.75 392 R392S 1.00 0.73 392 R392T 1.00 0.91 392 R392V 0.89 0.48 392 R392W 0.68 0.23 392 R392Y 1.00 0.60 393 D393A 0.98 0.77 393 D393C 0.69 0.48 393 D393E 0.92 0.81 393 D393F 0.84 0.61 393 D393G 1.08 0.75 393 D393H 0.88 0.75 393 D393I

393 D393K 1.09 0.80 393 D393L 1.04 0.70 393 D393N

393 D393P

393 D393Q 1.00 0.82 393 D393R 0.88 0.64 393 D393S 0.92 0.91 393 D393T 1.12 0.90 393 D393V 1.04 0.63 393 D393W 0.95 0.66 393 D393Y 1.01 0.66 394 Y394A 0.91 0.86 394 Y394D 0.98 0.84 394 Y394E 0.92 1.03 394 Y394F 1.07 0.98 394 Y394G 1.13 0.85 394 Y394H 1.04 0.99 394 Y394I 1.11 0.95 394 Y394K 1.09 1.07 394 Y394L 1.22 1.11 394 Y394M 0.74 0.23 394 Y394N 1.00 1.01 394 Y394P

394 Y394Q 1.09 1.13 394 Y394S 1.11 1.15 394 Y394V 3.00 0.75 394 Y394W 1.11 1.16 400 H400A 1.24 0.89 400 H400C 1.16 0.73 400 H400D 1.05 0.82 400 H400E 0.99 0.95 400 H400F 1.01 0.94 400 H400G 0.90 0.83 400 H400I 1.04 0.91 400 H400K 0.92 1.03 400 H400L 0.90 0.88 400 H400M 1.01 0.91 400 H400N 1.26 0.92 400 H400P

400 H400Q 0.96 0.94 400 H400R 1.03 0.87 400 H400S 0.94 0.92 400 H400T 0.95 0.88 400 H400V 1.28 0.91 400 H400W 1.17 0.80 400 H400Y 1.15 0.92 402 Y402A 1.07 0.97 402 Y402C 0.92 0.76 402 Y402D 0.90 0.80 402 Y402E 1.09 0.77 402 Y402F 0.89 0.82 402 Y402G 0.92 0.81 402 Y402H 1.21 0.91 402 Y402I 1.36 0.75 402 Y402K 0.95 0.84 402 Y402L 1.09 0.49 402 Y402M 1.14 0.88 402 Y402N 1.06 0.86 402 Y402P 1.03 0.28 402 Y402Q 0.98 0.83 402 Y402R 1.16 0.75 402 Y402T 1.32 1.02 402 Y402V 1.40 0.95 402 Y402W 1.24 0.89 403 L403A 1.20 0.89 403 L403C 1.10 0.98 403 L403D 1.03 0.95 403 L403E 1.26 0.93 403 L403F 1.03 0.74 403 L403G 1.22 0.96 403 L403H 1.10 0.90 403 L403M 1.11 0.99 403 L403N 0.98 0.95 403 L403P 0.78 0.47 403 L403Q 1.24 0.98 403 L403R 1.36 1.01 403 L403S 1.17 1.00 403 L403T 1.53 0.99 403 L403V 1.34 1.00 403 L403W 1.15 0.85 403 L403Y 1.16 0.97 404 D404A 1.12 0.73 404 D404C 1.28 0.61 404 D404E 1.38 0.78 404 D404G 1.25 0.77 404 D404I 1.20 0.84 404 D404K 1.10 0.83 404 D404L 1.09 0.91 404 D404M 1.13 0.76 404 D404N 1.13 0.98 404 D404P 1.05 0.56 404 D404Q 1.17 0.91 404 D404R 1.15 0.77 404 D404S 1.19 0.99 404 D404V 1.28 0.79 404 D404W 1.05 0.76 404 D404Y 1.08 0.81 406 S406A 0.99 0.99 406 S406C 1.11 0.85 406 S406D 0.93 1.02 406 S406E 0.95 0.91 406 S406F 0.86 0.88 406 S406G 0.93 0.86 406 S406H 0.88 0.98 406 S406I 0.92 0.91 406 S406K 0.95 0.82 406 S406L 0.94 0.98 406 S406M 0.89 0.90 406 S406N 1.09 0.94 406 S406P 0.91 0.93 406 S406Q

406 S406T 1.18 0.97 406 S406V 1.14 0.87 406 S406Y 0.99 0.80 407 D407C 1.14 0.41 407 D407E 0.82 0.59 407 D407F 0.88 0.35 407 D407G 1.10 0.38 407 D407H 0.85 0.63 407 D407I 1.05 0.22 407 D407K 1.00 0.44 407 D407L 0.91 0.18 407 D407M 1.05 0.37 407 D407N 1.11 0.96 407 D407P

407 D407Q 0.94 0.53 407 D407R 0.78 0.36 407 D407S 0.93 0.65 407 D407T 1.06 0.49 407 D407V 0.93 0.29 407 D407W 1.06 0.20 407 D407Y 0.85 0.38 410 G410A 0.90 1.00 410 G410C 1.04 0.81 410 G410D

410 G410E

410 G410F 0.96 0.22 410 G410H 0.93 0.34 410 G410I

410 G410L

410 G410M 1.13 0.35 410 G410N 0.99 0.27 410 G410P

410 G410Q 1.05 0.14 410 G410R 0.98 0.27 410 G410T 1.08 0.70 410 G410V 1.10 0.42 410 G410W

410 G410Y 0.92 0.49 413 R413A 1.02 1.06 413 R413D 0.71 0.40 413 R413E 0.86 0.67 413 R413G 1.19 0.33 413 R413H 1.06 0.95 413 R413I 0.96 0.75 413 R413K 1.08 0.95 413 R413L 1.02 0.96 413 R413M 0.81 0.81 413 R413N 0.93 0.72 413 R413P

413 R413Q 0.81 0.35 413 R413S 0.85 0.87 413 R413T

0.74 413 R413V 0.93 0.73 413 R413W 0.92 0.41 413 R413Y 0.73 0.49 414 E414A 1.06 0.70 414 E414C 1.05 0.55 414 E414D 1.13 0.75 414 E414F 0.81 0.59 414 E414G 0.82 0.68 414 E414H 0.89 0.65 414 E414I 0.98 0.60 414 E414K 0.96 0.65 414 E414L 1.16 0.71 414 E414M 0.88 0.72 414 E414N 0.99 0.57 414 E414P 0.85 0.60 414 E414Q 0.85 0.70 414 E414R 1.00 0.65 414 E414S 0.91 0.63 414 E414T 0.79 0.67 414 E414W 1.03 0.25 414 E414Y 0.78 0.58 416 V416A 0.93 0.67 416 V416C 0.94 0.61 416 V416D 1.05 0.71 416 V416F

416 V416H 0.92 0.78 416 V416I 0.83 0.74 416 V416K 0.71 0.65 416 V416L 0.96 0.81 416 V416M 1.06 0.78 416 V416N 0.92 0.66 416 V416P 1.18 0.53 416 V416Q 1.02 0.74 416 V416R 1.02 0.29 416 V416S 1.15 0.46 416 V416T 1.01 0.65 416 V416W 0.83 0.55 416 V416Y 0.89 0.69 419 K419A 1.36 1.29 419 K419C 1.24 0.67 419 K419D 1.00 0.94 419 K419E 0.87 0.65 419 K419F 0.90 0.17 419 K419H

0.05 419 K419I 1.12 1.32 419 K419L

419 K419M 1.20 1.27 419 K419N

419 K419P 1.11 1.23 419 K419Q 0.96 0.61 419 K419R

419 K419S 1.13 1.51 419 K419T 1.42 1.05 419 K419V 1.14 1.55 419 K419W 0.98 0.14 419 K419Y 1.15 0.11 422 S422A 0.64 0.97 422 S422C 0.96 0.71 422 S422D 0.97 0.96 422 S422E 1.31 0.78 422 S422F 0.96 0.71 422 S422G 1.20 0.99 422 S422H 1.06 0.66 422 S422I 1.11 0.85 422 S422K 1.16 0.96 422 S422L 0.99 0.74 422 S422M 1.04 0.94 422 S422N 1.12 1.03 422 S422P 0.84 0.70 422 S422Q 0.15 0.82 422 S422R 1.02 0.94 422 S422T 0.97 0.92 422 S422V 1.17 0.88 422 S422W 0.96 0.70 422 S422Y 1.09 0.92 427 L427A 0.93 0.66 427 L427C 1.02 0.68 427 L427D

427 L427E 0.86 0.27 427 L427F 0.89 0.30 427 L427G 0.63 0.26 427 L427H

427 L427I 1.08 0.64 427 L427K

427 L427M 0.86 0.79 427 L427N 0.76 0.31 427 L427P 1.13 0.06 427 L427Q 0.95 0.53 427 L427R

427 L427S 0.78 0.27 427 L427T 0.80 0.70 427 L427V 0.82 0.72 427 L427W

427 L427Y

433 G433A 1.27 1.08 433 G433C 1.15 0.69 433 G433D 1.05 0.96 433 G433E 0.92 0.99 433 G433F 1.04 0.92 433 G433H 1.27 0.99 433 G433I 1.37 0.86 433 G433K 1.27 1.05 433 G433L 1.30 0.90 433 G433M 1.23 1.01 433 G433N 1.07 0.75 433 G433P 1.13 0.95 433 G433Q 0.78 0.99 433 G433R 1.00 0.91 433 G433S 1.17 0.96 433 G433T 1.17 0.90 433 G433V 1.27 0.95 433 G433Y 1.26 1.01 436 K436A 0.92 0.94 436 K436C 0.90 0.84 436 K436D 0.86 0.93 436 K436E 0.70 0.87 436 K436F 0.81 0.64 436 K436G 0.84 0.77 436 K436H 1.09 0.89 436 K436I 1.08 0.81 436 K436L 1.01 0.78 436 K436M 0.76 0.85 436 K436N 0.98 0.92 436 K436P 0.88 0.71 436 K436Q 1.01 0.96 436 K436R 1.06 0.79 436 K436S 0.75 0.92 436 K436T 0.95 0.90 436 K436V 0.98 0.87 436 K436W 1.07 0.71 436 K436Y 0.99 0.80 439 Y439A 1.02 0.78 439 Y439D 1.01 0.85 439 Y439E

439 Y439F 0.77 0.78 439 Y439G 1.01 0.77 439 Y439H 0.96 0.73 439 Y439I

439 Y439K 0.96 0.74 439 Y439L

439 Y439M 1.04 0.77 439 Y439N 0.96 0.83 439 Y439P 0.87 0.85 439 Y439Q 0.90 0.88 439 Y439R 0.75 0.80 439 Y439S 0.94 0.82 439 Y439T 0.84 0.79 439 Y439V 1.04 0.70 439 Y439W 0.86 0.72 442 K442A 1.38 0.98 442 K442C

442 K442F 1.04 0.97 442 K442G 1.23 1.02 442 K442H 1.07 1.04 442 K442I 1.13 0.93 442 K442N 1.39 1.03 442 K442P 1.11 1.03 442 K442Q 1.11 1.05 442 K442R 1.33 1.01 442 K442S 1.24 1.07 442 K442T 1.34 1.06 442 K442V 1.20 0.99 442 K442W 1.32 0.98 442 K442Y 1.24 1.08 445 A445C 0.98 0.83 445 A445D 1.04 0.87 445 A445G 1.21 1.01 445 A445H 0.90 0.93 445 A445I 1.25 0.84 445 A445K 1.20 0.11 445 A445L 1.17 0.92 445 A445N 1.20 0.91 445 A445P 0.91 0.77 445 A445Q

445 A445R 0.91 0.89 445 A445S 1.16 0.94 445 A445T 1.29 0.88 445 A445V 1.27 0.93 445 A445W 1.25 0.80 447 K447A 1.09 1.06 447 K447C 1.11 0.87 447 K447D 1.00 0.99 447 K447F 1.09 0.84 447 K447G 1.06 0.94 447 K447H 1.13 0.92 447 K447I 1.22 0.91 447 K447L 1.06 1.01 447 K447M 1.07 0.96 447 K447N 1.43 0.97 447 K447Q 1.34 1.00 447 K447R 1.10 0.96 447 K447S 0.90 0.92 447 K447T 1.21 0.37 447 K447V 0.69 0.86 447 K447W 1.31 0.89 447 K447Y 1.21 0.96 448 V448A 0.98 0.96 448 V448C 1.36 0.98 448 V448D 1.15 1.02 448 V448E

448 V448F 1.48 1.01 448 V448G 1.26 1.05 448 V448H 1.37 1.03 448 V448I 1.44 0.97 448 V448K 1.20 1.07 448 V448L 1.04 1.08 448 V448M 1.13 0.97 448 V448N 1.24 0.70 448 V448P 0.84 1.19 448 V448Q 1.18 1.16 448 V448R

448 V448S 1.20 1.10 448 V448T

448 V448W 1.08 0.89 448 V448Y 1.33 1.27 450 Y450A 0.95 0.94 450 Y450C 1.22 0.84 450 Y450D 1.19 0.95 450 Y450E 1.01 0.92 450 Y450G 1.02 0.93 450 Y450H 1.23 0.90 450 Y450K 1.18 0.94 450 Y450L 0.93 0.69 450 Y450M 1.29 0.89 450 Y450N 1.23 0.96 450 Y450P 0.75 0.30 450 Y450Q 1.00 0.95 450 Y450R 1.22 1.02 450 Y450S 1.22 1.01 450 Y450T 1.32 0.96 450 Y450V

450 Y450W 1.21 0.95 452 L452A 1.08 1.06 452 L452C 1.00 1.01 452 L452D 0.98 1.08 452 L452E 0.75 0.55 452 L452F 0.79 0.93 452 L452G 1.07 1.00 452 L452H 1.05 0.99 452 L452K 1.11 1.08 452 L452M 1.13 1.09 452 L452N 1.06 1.28 452 L452P 1.02 0.78 452 L452Q 0.92 1.22 452 L452R 0.93 1.26 452 L452S 0.86 1.21 452 L452T 1.02 1.18 452 L452V 1.14 1.14 452 L452Y 1.17 1.07 455 N455A 1.07 1.04 455 N455C 0.85 0.89 455 N455D 1.07 0.97 455 N455E 1.14 0.94 455 N455G 1.23 1.00 455 N455H 1.05 1.01 455 N455I 1.23 0.95 455 N455K 1.10 1.08 455 N455L 1.06 0.97 455 N455M 0.95 0.96 455 N455P 1.36 0.93 455 N455Q 0.96 0.91 455 N455R 1.13 1.02 455 N455S 1.04 0.91 455 N455T 1.16 0.90 455 N455V 1.26 0.89 455 N455W 1.12 0.76 455 N455Y 1.08 0.15 463 N463A 1.25 1.06 463 N463D 0.97 1.02 463 N463F 1.04 0.87 463 N463G 1.04 1.00 463 N463H 1.12 0.99 463 N463I

463 N463K 1.07 1.00 463 N463L 1.16 1.01 463 N463M 1.24 1.08 463 N463P 0.93 1.05 463 N463Q 0.98 1.04 463 N463R 0.95 0.93 463 N463S 1.27 0.96 463 N463T 1.38 0.91 463 N463V 1.32 0.86 463 N463W 1.45 0.74 463 N463Y 1.20 0.90 465 D465A 0.76 1.06 465 D465C 0.84 0.74 465 D465E 0.95 0.93 465 D465F 0.78 0.89 465 D465G 1.35 0.92 465 D465H 1.06 0.92 465 D465I 1.37 0.85 465 D465K 1.53 0.88 465 D465L 1.14 0.95 465 D465M 1.06 0.98 465 D465N 1.32 0.93 465 D465P 1.13 0.71 465 D465Q 0.86 0.94 465 D465R 1.18 0.90 465 D465S 0.87 0.98 465 D465T 1.42 0.92 465 D465V 1.24 0.93 465 D465W 1.00 0.83 465 D465Y 1.06 0.93 469 E469A 1.16 1.01 469 E469C 1.03 0.86 469 E469D 1.22 1.02 469 E469F 1.11 1.00 469 E469G 1.19 1.00 469 E469H 1.04 0.96 469 E469K 1.16 0.96 469 E469L 1.10 0.98 469 E469M

469 E469N 1.19 0.47 469 E469P 0.85 1.05 469 E469Q 1.03 1.04 469 E469R 1.01 0.75 469 E469S 0.91 1.08 469 E469T 1.15 1.06 469 E469V 1.15 1.08 469 E469W 1.24 0.97 469 E469Y 1.35 1.09 471 K471A 1.09 1.09 471 K471C 1.04 0.91 471 K471D 1.01 1.06 471 K471F 1.10 1.05 471 K471G 1.13 1.12 471 K471H 1.00 1.10 471 K471I 1.22 1.02 471 K471L 0.99 1.07 471 K471M 0.95 1.14 471 K471N 1.04 1.12 471 K471P 0.84 0.98 471 K471Q 0.90 1.08 471 K471R 0.77 1.33 471 K471S 0.97 1.01 471 K471T 1.11 1.09 471 K471V 1.28 1.11 471 K471Y 1.15 1.36 473 N473A 1.03 0.99 473 N473C 1.15 0.74 473 N473D 1.14 0.98 473 N473E 1.20 0.99 473 N473F 1.10 0.83 473 N473G 1.35 0.99 473 N473H 1.02 0.91 473 N473I 0.66 0.45 473 N473K 1.02 1.02 473 N473L

0.97 473 N473M 1.11 1.00 473 N473P 1.01 0.95 473 N473Q 1.13 0.99 473 N473R 1.08 1.05 473 N473S 1.15 0.98 473 N473T 1.04 1.04 473 N473V

473 N473W 0.85 0.64 473 N473Y 1.23 0.86 476 S476A 1.51 1.02 476 S476C 0.91 0.89 476 S476D 0.98 0.91 476 S476E 1.08 0.91 476 S476F 1.09 0.87 476 S476G 1.22 0.97 476 S476H 1.07 0.96 476 S476I 1.03 0.78 476 S476K 1.01 0.97 476 S476L 1.46 0.93 476 S476M 1.58 1.08 476 S476N 1.61 0.98 476 S476P 1.02 0.62 476 S476Q 1.13 1.03 476 S476R 1.01 1.08 476 S476T 1.78 1.01 476 S476V 1.21 0.89 476 S476W 1.43 0.78 476 S476Y 1.79 0.94

Table 26-5 lists AmyS variants which are combinable mutations (2,250) for the 152 positions. These variants have Performance index values>0.5 for at least one property (activity or stability) and >0.05 for both properties.

TABLE 26-5 Combinable mutations in AmyS Stability Activity POS variant PI PI 5 N005A 0.95 0.32 5 N005C 0.98 0.29 5 N005E 1.04 0.43 5 N005F 0.79 0.15 5 N005G 0.88 0.34 5 N005H 0.89 0.43 5 N005I 1.00 0.10 5 N005K 0.90 0.34 5 N005L 1.04 0.10 5 N005M 0.84 0.18 5 N005P 1.10 0.40 5 N005Q 1.07 0.58 5 N005R 0.94 0.40 5 N005S 0.98 0.35 5 N005T 0.83 0.35 5 N005V 0.88 0.16 5 N005W 0.94 0.07 5 N005Y 1.07 0.21 6 G006A 1.10 1.59 6 G006D 1.14 1.64 6 G006E 1.08 1.93 6 G006H 0.95 2.29 6 G006I 1.23 1.47 6 G006K 0.93 2.36 6 G006L 1.15 1.61 6 G006M 1.11 1.60 6 G006N 1.21 1.61 6 G006P 1.10 2.47 6 G006Q 1.26 1.34 6 G006R 0.98 1.28 6 G006S 1.12 1.86 6 G006T 1.21 2.01 6 G006V 1.29 1.54 6 G006W 1.13 1.32 6 G006Y 1.07 1.88 13 E013A 0.32 1.01 13 E013C 0.22 0.68 13 E013D 0.08 1.03 13 E013G 0.18 1.00 13 E013H 0.60 1.10 13 E013I 0.15 0.87 13 E013K 0.22 1.22 13 E013L 0.20 1.02 13 E013M 0.20 0.96 13 E013Q 0.21 0.96 13 E013R 0.28 1.04 13 E013S 0.28 0.92 13 E013T 0.19 0.79 13 E013V 0.19 0.76 13 E013W 0.05 0.76 13 E013Y 0.89 0.93 14 W014A 0.95 0.77 14 W014C 0.91 0.71 14 W014D 0.81 0.59 14 W014E 0.95 1.07 14 W014F 1.06 1.25 14 W014G 0.97 0.88 14 W014I 1.12 0.40 14 W014K 1.01 0.69 14 W014L 0.88 0.15 14 W014M 1.18 0.84 14 W014N 0.92 0.99 14 W014P 0.84 0.98 14 W014Q 0.94 0.67 14 W014R 0.97 0.67 14 W014S 0.97 1.02 14 W014T 1.22 1.22 14 W014V 1.17 0.81 14 W014Y 1.08 1.71 15 Y015A 1.05 1.48 15 Y015C 0.70 1.15 15 Y015D 0.77 1.82 15 Y015E 0.68 1.96 15 Y015G 0.69 1.89 15 Y015H 1.01 1.85 15 Y015I 0.63 0.91 15 Y015K 0.74 1.58 15 Y015L 0.67 0.76 15 Y015M 0.72 1.12 15 Y015N 0.99 1.88 15 Y015P 0.57 1.59 15 Y015Q 0.80 1.74 15 Y015R 0.72 1.60 15 Y015S 0.58 1.78 15 Y015T 0.87 1.47 15 Y015W 0.95 1.44 16 L016A 0.81 1.31 16 L016D 0.93 1.12 16 L016E 1.09 1.21 16 L016F 2.17 0.98 16 L016G 0.61 1.35 16 L016H 0.96 1.21 16 L016I 0.79 1.12 16 L016K 0.79 1.41 16 L016M 0.94 1.15 16 L016N 0.92 1.32 16 L016P 0.35 1.30 16 L016Q 0.96 1.33 16 L016R 0.71 1.28 16 L016S 0.94 1.19 16 L016T 0.87 1.32 16 L016V 0.87 1.16 16 L016W 0.75 0.99 16 L016Y 0.97 1.10 18 D018A 1.08 0.89 18 D018F 0.68 0.58 18 D018G 0.88 0.87 18 D018H 0.84 0.84 18 D018I 0.79 0.70 18 D018K 0.88 0.65 18 D018L 0.60 0.72 18 D018N 0.73 1.01 18 D018P 0.84 1.04 18 D018Q 0.80 1.00 18 D018R 0.81 0.65 18 D018S 0.81 0.93 18 D018T 0.81 0.91 18 D018V 0.89 0.77 18 D018W 0.72 0.51 18 D018Y 0.72 0.87 20 G020A 0.79 0.25 20 G020C 0.58 0.24 20 G020D 0.92 0.96 20 G020E 0.89 0.95 20 G020F 0.65 0.13 20 G020H 0.75 0.11 20 G020I 0.96 0.28 20 G020M 0.69 0.10 20 G020N 0.78 0.09 20 G020Q 0.61 0.07 20 G020T 0.82 0.09 20 G020V 0.77 0.19 20 G020W 0.80 0.69 25 K025A 1.22 0.82 25 K025C 1.33 1.46 25 K025D 1.06 1.03 25 K025E 1.07 0.95 25 K025F 1.00 0.58 25 K025G 1.27 0.97 25 K025H 1.03 1.06 25 K025L 1.12 0.64 25 K025M 1.03 0.61 25 K025N 0.91 1.06 25 K025P 0.98 0.55 25 K025Q 1.24 1.07 25 K025R 1.08 0.96 25 K025S 1.07 0.98 25 K025T 1.14 0.89 25 K025Y 0.98 0.65 27 A027C 0.79 0.55 27 A027D 1.01 0.95 27 A027E 0.93 0.95 27 A027F 0.88 0.85 27 A027G 1.20 0.98 27 A027H 1.05 1.00 27 A027I 1.05 0.87 27 A027K 0.86 1.01 27 A027L 1.06 0.86 27 A027M 1.21 0.88 27 A027N 1.06 1.00 27 A027P 1.13 0.43 27 A027Q 1.00 0.96 27 A027R 1.11 0.89 27 A027S 1.16 0.97 27 A027T 1.20 0.90 27 A027V 1.20 0.82 27 A027W 1.13 0.76 27 A027Y 0.97 0.28 29 E029A 1.05 0.50 29 E029D 0.94 1.11 29 E029G 0.75 0.37 29 E029H 0.83 0.83 29 E029K 1.05 0.89 29 E029L 0.76 0.22 29 E029M 0.76 0.15 29 E029N 1.02 0.89 29 E029P 0.87 0.33 29 E029Q 1.04 0.86 29 E029R 1.09 0.92 29 E029S 0.97 0.83 29 E029T 0.95 0.59 29 E029W 0.74 0.10 36 L036A 0.95 0.85 36 L036C 0.83 0.43 36 L036D 0.91 0.27 36 L036E 0.90 0.40 36 L036F 1.14 0.90 36 L036G 0.92 0.34 36 L036H 0.92 0.77 36 L036I 1.17 0.89 36 L036K 1.01 1.05 36 L036M 1.05 1.05 36 L036N 1.02 0.68 36 L036P 0.90 0.06 36 L036Q 1.40 0.78 36 L036R 1.12 0.76 36 L036S 1.25 0.69 36 L036T 1.11 0.64 36 L036V 0.88 0.97 36 L036W 0.92 0.63 36 L036Y 1.07 0.91 39 T039C 1.09 1.05 39 T039D 1.15 1.47 39 T039E 1.15 1.32 39 T039F 1.16 0.48 39 T039G 1.23 1.05 39 T039H 1.16 1.10 39 T039K 1.12 1.10 39 T039M 1.18 0.54 39 T039N 1.14 1.64 39 T039P 1.11 0.26 39 T039Q 1.20 1.43 39 T039R 1.01 1.10 39 T039S 1.15 1.02 39 T039V 1.30 0.82 39 T039W 1.11 0.25 50 T050A 1.09 0.98 50 T050C 1.03 0.34 50 T050D 0.87 0.91 50 T050F 0.86 0.43 50 T050G 1.00 1.18 50 T050H 0.97 0.82 50 T050I 1.24 0.61 50 T050K 1.13 0.80 50 T050L 1.22 0.67 50 T050M 1.32 0.62 50 T050N 1.20 1.12 50 T050P 1.03 0.99 50 T050Q 1.31 1.08 50 T050R 1.13 0.79 50 T050S 1.07 1.09 50 T050V 1.02 0.79 50 T050W 0.90 0.18 50 T050Y 1.14 0.42 52 R052A 0.99 1.02 52 R052C 0.87 0.62 52 R052D 0.76 0.85 52 R052E 0.77 0.97 52 R052G 0.96 0.93 52 R052H 0.91 0.99 52 R052K 0.93 1.02 52 R052L 1.10 0.98 52 R052M 1.01 1.00 52 R052N 0.95 0.99 52 R052P 1.05 0.95 52 R052S 1.21 0.92 52 R052T 1.11 1.00 52 R052V 1.14 0.95 52 R052W 1.00 0.83 52 R052Y 0.99 0.96 53 S053A 1.03 1.00 53 S053C 0.73 0.58 53 S053D 0.75 0.83 53 S053E 1.05 0.88 53 S053F 0.87 0.85 53 S053G 1.14 0.93 53 S053H 1.12 1.00 53 S053I 0.99 1.12 53 S053K 1.03 1.10 53 S053L 0.93 0.96 53 S053M 0.96 0.97 53 S053P 0.88 1.00 53 S053Q 0.94 0.94 53 S053R 0.83 1.15 53 S053T 1.25 1.02 53 S053V 1.11 0.94 53 S053W 1.09 0.84 53 S053Y 0.94 0.93 54 D054A 0.34 0.88 54 D054C 0.64 0.38 54 D054G 0.11 0.97 54 D054H 0.11 1.04 54 D054I 0.30 0.83 54 D054M 0.11 0.88 54 D054N 0.94 1.05 54 D054R 0.06 0.89 54 D054S 0.38 0.96 54 D054T 0.17 0.95 54 D054V 0.17 0.77 67 E067C 1.08 0.75 67 E067D 0.90 1.07 67 E067G 1.01 1.13 67 E067H 1.04 1.03 67 E067K 0.98 0.94 67 E067L 0.97 0.95 67 E067M 0.93 0.91 67 E067N 1.32 0.95 67 E067Q 0.93 0.95 67 E067R 1.01 0.90 67 E067S 1.23 1.00 67 E067T 0.99 0.98 67 E067Y 1.11 0.93 71 K071A 0.72 0.81 71 K071C 0.80 0.61 71 K071D 0.69 0.71 71 K071E 0.80 0.84 71 K071F 0.47 0.61 71 K071G 0.74 0.91 71 K071H 0.96 0.88 71 K071I 0.83 0.75 71 K071L 0.55 0.61 71 K071M 0.80 0.68 71 K071N 1.11 0.89 71 K071P 0.92 0.86 71 K071Q 0.98 0.77 71 K071R 1.10 1.10 71 K071S 0.99 0.97 71 K071T 0.95 0.83 71 K071V 0.94 0.84 71 K071W 0.82 0.91 71 K071Y 0.52 0.71 73 T073A 0.97 1.11 73 T073C 0.91 0.60 73 T073D 0.89 1.02 73 T073E 0.75 1.08 73 T073F 0.73 0.99 73 T073G 0.79 1.12 73 T073H 0.86 0.88 73 T073I 0.66 1.02 73 T073K 0.20 0.97 73 T073L 0.47 1.17 73 T073M 0.59 0.64 73 T073N 0.73 1.08 73 T073P 0.57 0.98 73 T073R 0.40 1.11 73 T073S 0.87 1.10 73 T073V 0.67 1.09 73 T073W 0.83 1.07 73 T073Y 0.79 1.10 75 R075A 1.05 1.14 75 R075C 0.88 0.85 75 R075D 0.87 0.99 75 R075E 0.86 1.01 75 R075F 0.76 0.92 75 R075G 0.79 1.04 75 R075H 0.85 1.07 75 R075I 0.86 1.01 75 R075L 0.88 1.04 75 R075M 1.04 1.04 75 R075P 0.90 0.93 75 R075Q 0.90 0.95 75 R075S 0.66 0.60 75 R075T 0.98 0.88 75 R075V 0.78 0.94 75 R075W 0.75 0.93 75 R075Y 0.68 1.04 77 K077A 0.38 0.98 77 K077C 0.28 0.51 77 K077E 0.11 0.77 77 K077F 0.20 0.72 77 K077G 0.13 0.76 77 K077I 0.16 1.00 77 K077L 0.54 0.98 77 K077M 0.58 0.99 77 K077Q 0.07 0.86 77 K077R 0.77 1.07 77 K077S 0.11 0.89 77 K077V 0.05 0.83 80 T080A 0.88 1.01 80 T080C 0.91 0.69 80 T080D 1.22 0.86 80 T080E 0.71 0.92 80 T080F 1.10 0.50 80 T080G 1.02 0.93 80 T080H 1.01 0.95 80 T080I 1.29 0.82 80 T080K 0.90 0.86 80 T080L 0.82 0.98 80 T080M 0.97 0.95 80 T080N 0.90 1.00 80 T080P 0.88 0.88 80 T080Q 0.87 0.88 80 T080R 0.99 0.76 80 T080S 0.83 1.09 80 T080V 0.87 0.87 80 T080W 0.77 0.89 80 T080Y 0.72 0.97 81 K081A 0.87 0.94 81 K081C 0.84 0.74 81 K081D 0.96 0.83 81 K081E 0.69 0.92 81 K081G 0.86 0.81 81 K081H 0.73 1.03 81 K081I 0.82 0.79 81 K081L 0.87 1.01 81 K081M 0.93 1.04 81 K081P 0.90 0.79 81 K081Q 0.84 1.03 81 K081R 0.90 1.04 81 K081S 0.74 0.98 81 K081T 0.80 0.93 81 K081V 0.66 1.03 81 K081W 0.60 0.98 81 K081Y 0.89 1.20 83 Q083A 1.20 0.98 83 Q083C 1.79 0.17 83 Q083D 0.94 0.92 83 Q083E 0.98 0.95 83 Q083F 0.87 0.80 83 Q083G 0.76 1.01 83 Q083H 0.78 0.86 83 Q083I 0.69 0.85 83 Q083L 0.77 0.91 83 Q083M 0.91 0.96 83 Q083P 1.01 0.82 83 Q083R 0.91 0.90 83 Q083S 0.75 0.99 83 Q083T 0.84 0.84 83 Q083V 0.73 0.80 83 Q083W 0.82 0.78 83 Q083Y 0.71 0.93 85 L085A 0.94 1.06 85 L085C 0.90 0.63 85 L085D 0.84 1.04 85 L085E 1.09 1.02 85 L085G 0.85 0.90 85 L085H 0.73 1.02 85 L085I 0.89 0.88 85 L085K 0.96 0.93 85 L085M 1.01 1.04 85 L085N 1.10 0.89 85 L085P 1.01 0.72 85 L085Q 0.91 0.99 85 L085R 0.96 1.01 85 L085S 1.02 1.04 85 L085T 0.83 1.12 85 L085W 0.93 0.95 85 L085Y 0.70 1.08 90 A090C 1.00 0.65 90 A090D 1.12 0.92 90 A090E 1.20 0.92 90 A090F 0.99 0.76 90 A090G 1.04 0.87 90 A090H 1.05 1.03 90 A090I 0.90 0.83 90 A090K 0.93 1.04 90 A090L 0.76 0.92 90 A090M 1.02 1.02 90 A090N 1.02 0.98 90 A090P 1.39 0.10 90 A090Q 0.94 0.93 90 A090R 0.90 0.90 90 A090S 1.16 0.99 90 A090T 0.78 0.88 90 A090V 0.79 0.87 90 A090W 0.69 0.84 90 A090Y 0.83 0.96 92 H092C 0.75 0.29 92 H092D 1.06 0.69 92 H092E 0.88 0.76 92 H092F 0.92 0.28 92 H092G 0.86 0.81 92 H092K 0.89 0.98 92 H092N 0.85 0.78 92 H092Q 0.80 0.89 92 H092R 0.75 0.96 92 H092S 0.70 0.87 92 H092T 0.68 0.47 92 H092V 0.70 0.28 92 H092W 0.83 0.44 92 H092Y 0.71 0.63 106 H106D 0.58 0.07 106 H106P 0.59 0.06 107 K107A 0.46 0.81 107 K107C 0.42 0.67 107 K107D 0.32 0.51 107 K107E 0.35 0.70 107 K107F 0.42 0.66 107 K107G 0.23 0.76 107 K107H 0.34 0.94 107 K107I 0.29 0.69 107 K107L 0.53 0.75 107 K107M 0.60 0.79 107 K107N 0.43 0.88 107 K107Q 0.63 0.74 107 K107R 1.05 0.71 107 K107S 0.30 0.78 107 K107T 0.38 0.72 107 K107V 0.41 0.70 107 K107Y 0.40 0.64 111 D111A 0.55 0.95 111 D111C 0.71 0.60 111 D111E 0.87 1.01 111 D111F 0.63 0.65 111 D111G 0.74 0.90 111 D111H 0.50 0.85 111 D111I 0.56 0.91 111 D111K 0.45 0.62 111 D111L 0.44 0.86 111 D111M 0.65 1.00 111 D111N 0.97 0.87 111 D111P 0.78 0.71 111 D111Q 0.77 0.95 111 D111R 0.53 0.07 111 D111S 0.67 0.91 111 D111T 0.61 1.02 111 D111V 0.58 1.02 111 D111W 0.42 0.54 111 D111Y 0.49 0.92 113 T113A 0.89 0.97 113 T113C 0.80 0.82 113 T113D 0.94 0.95 113 T113E 0.92 0.91 113 T113F 0.76 0.92 113 T113G 0.88 1.08 113 T113H 0.88 0.96 113 T113I 1.14 0.88 113 T113K 0.93 1.13 113 T113L 1.08 1.08 113 T113M 0.83 0.99 113 T113P 1.05 0.96 113 T113Q 0.88 1.05 113 T113R 0.88 1.03 113 T113V 1.12 0.94 113 T113W 1.06 0.88 114 E114A 0.54 0.97 114 E114C 0.62 0.76 114 E114D 0.71 0.82 114 E114F 0.36 0.92 114 E114G 0.59 1.01 114 E114H 0.49 0.92 114 E114I 0.54 0.86 114 E114L 0.43 0.97 114 E114M 0.77 0.97 114 E114N 0.67 0.88 114 E114R 0.35 0.84 114 E114T 0.54 0.94 114 E114V 0.43 0.85 114 E114W 0.31 0.94 114 E114Y 0.26 0.93 120 E120A 0.29 1.20 120 E120C 0.24 0.89 120 E120H 0.09 0.90 120 E120I 0.60 0.87 120 E120L 0.20 0.97 120 E120M 0.39 0.96 120 E120N 0.16 1.02 120 E120Q 0.66 1.10 120 E120R 0.12 1.12 120 E120S 0.08 1.07 120 E120T 0.22 1.06 120 E120V 0.53 0.93 120 E120W 0.15 0.81 120 E120Y 0.07 0.98 121 V121C 0.92 0.55 121 V121T 0.07 0.92 128 Q128C 0.42 0.95 128 Q128D 0.15 1.05 128 Q128E 0.90 1.00 128 Q128H 0.34 1.05 128 Q128I 0.90 0.89 128 Q128K 0.52 1.15 128 Q128L 0.47 0.97 128 Q128N 0.12 1.05 128 Q128R 0.31 1.14 128 Q128S 0.28 1.02 128 Q128V 0.86 0.97 128 Q128W 0.07 0.76 128 Q128Y 0.13 0.86 131 S131D 0.26 1.08 131 S131G 0.24 0.86 131 S131N 0.76 1.02 131 S131T 0.49 0.90 133 T133A 0.95 1.13 133 T133C 0.49 0.97 133 T133D 1.03 0.99 133 T133E 0.82 1.02 133 T133F 0.17 0.97 133 T133G 0.47 0.84 133 T133H 0.41 1.19 133 T133I 0.86 0.96 133 T133K 0.47 0.85 133 T133L 0.41 1.06 133 T133M 0.51 1.05 133 T133N 0.68 1.13 133 T133P 1.41 1.08 133 T133Q 0.63 1.10 133 T133R 0.18 1.13 133 T133S 0.72 1.08 133 T133V 1.25 0.92 133 T133W 0.14 0.98 133 T133Y 0.41 1.01 137 Q137A 0.92 0.97 137 Q137C 1.09 0.77 137 Q137D 0.89 0.96 137 Q137E 1.06 0.87 137 Q137F 0.85 0.86 137 Q137G 1.13 0.94 137 Q137H 0.95 1.05 137 Q137I 0.93 0.22 137 Q137L 1.20 0.82 137 Q137M 1.30 0.83 137 Q137P 0.07 1.05 137 Q137R 0.95 1.05 137 Q137S 1.45 0.98 137 Q137T 1.12 0.91 137 Q137V 1.02 0.86 137 Q137W 1.06 0.88 137 Q137Y 0.94 0.89 138 A138G 0.90 1.02 138 A138I 0.23 0.90 138 A138N 0.50 0.94 138 A138P 1.07 1.15 138 A138Q 0.13 0.69 138 A138S 1.12 1.02 138 A138T 1.16 1.05 138 A138V 1.17 0.87 138 A138Y 0.14 0.97 139 W139A 0.82 0.89 139 W139C 0.75 0.39 139 W139D 0.93 1.40 139 W139E 0.81 0.97 139 W139G 0.79 0.74 139 W139H 0.97 1.59 139 W139I 0.74 0.58 139 W139K 0.68 0.42 139 W139L 0.78 0.59 139 W139M 0.87 1.00 139 W139N 1.13 0.85 139 W139Q 0.82 0.79 139 W139R 0.96 1.29 139 W139S 0.93 1.04 139 W139T 0.71 0.87 139 W139V 0.72 0.66 139 W139Y 1.14 1.63 141 K141A 1.09 0.73 141 K141C 1.03 0.85 141 K141D 0.89 0.98 141 K141E 3.48 0.92 141 K141F 0.89 0.80 141 K141G 1.18 0.96 141 K141H 1.13 0.99 141 K141I 1.40 0.87 141 K141L 1.22 0.85 141 K141M 1.23 1.01 141 K141N 1.11 1.02 141 K141P 1.07 0.96 141 K141Q 1.28 0.97 141 K141R 1.23 0.99 141 K141S 1.21 0.98 141 K141T 1.17 0.94 141 K141V 1.21 1.00 141 K141W 1.16 0.87 141 K141Y 1.17 0.88 143 D143A 0.95 1.04 143 D143C 1.11 0.84 143 D143E 1.12 0.98 143 D143G 1.13 1.09 143 D143H 0.91 0.98 143 D143I 1.05 0.94 143 D143K 0.86 0.96 143 D143M 0.86 1.05 143 D143N 1.10 0.99 143 D143P 0.98 0.84 143 D143V 1.00 1.01 143 D143W 1.00 0.99 143 D143Y 0.75 0.15 147 R147A 0.73 0.25 147 R147D 0.66 0.07 147 R147G 0.74 0.11 147 R147H 0.81 0.21 147 R147K 1.05 0.48 147 R147M 0.65 0.07 147 R147N 0.91 0.30 147 R147Q 0.88 0.30 147 R147S 0.90 0.39 147 R147T 0.90 0.10 149 N149A 0.94 0.93 149 N149D 0.89 0.95 149 N149E 0.98 0.93 149 N149F 1.09 0.85 149 N149G 0.90 0.93 149 N149H 1.01 0.98 149 N149I 1.15 0.83 149 N149K 0.90 0.88 149 N149L 0.88 0.94 149 N149Q 1.00 0.93 149 N149R 0.80 0.95 149 N149S 0.94 1.03 149 N149V 1.06 0.87 149 N149W 1.01 0.87 150 T150A 0.90 0.96 150 T150C 1.03 0.72 150 T150D 0.82 0.87 150 T150E 4.54 0.87 150 T150G 0.99 0.86 150 T150I 0.82 0.93 150 T150K 0.86 0.96 150 T150L 0.83 0.07 150 T150M 1.05 1.00 150 T150N 0.98 1.08 150 T150Q 0.83 0.99 150 T150R 0.99 1.04 150 T150S 0.77 0.96 150 T150V 0.90 0.93 150 T150Y 1.18 1.00 151 Y151A 0.96 0.87 151 Y151C 0.80 0.67 151 Y151D 0.99 0.71 151 Y151E 0.76 0.71 151 Y151F 0.96 0.88 151 Y151G 1.17 0.79 151 Y151H 1.04 0.87 151 Y151I 1.22 0.78 151 Y151L 1.05 0.90 151 Y151M 1.02 0.83 151 Y151N 0.98 0.91 151 Y151P 0.89 0.77 151 Y151Q 1.07 0.75 151 Y151R 1.05 0.76 151 Y151S 0.85 0.80 151 Y151T 1.04 0.80 151 Y151V 1.14 0.80 151 Y151W 1.16 0.79 152 S152A 0.95 0.88 152 S152C 0.83 0.75 152 S152E 1.09 0.71 152 S152F 0.75 0.22 152 S152G 1.25 0.91 152 S152H 0.99 0.71 152 S152I 0.81 0.22 152 S152K 0.74 0.58 152 S152N 1.20 0.43 152 S152Q 0.71 0.21 152 S152R 0.89 0.86 152 S152T 1.16 0.99 152 S152V 0.79 0.42 152 S152W 0.73 0.22 152 S152Y 0.91 0.26 155 K155A 1.10 0.85 155 K155C 0.92 0.72 155 K155D 0.94 0.85 155 K155E 0.82 0.79 155 K155G 1.05 0.58 155 K155H 1.04 0.84 155 K155L 1.05 0.89 155 K155M 0.91 0.91 155 K155N 1.18 0.90 155 K155P 0.99 0.94 155 K155Q 0.84 0.90 155 K155R 1.20 0.93 155 K155S 1.22 0.85 155 K155T 1.12 0.76 155 K155V 1.01 0.85 155 K155W 1.09 0.88 155 K155Y 1.21 0.80 160 H160A 0.89 0.89 160 H160C 0.84 0.98 160 H160D 0.89 0.69 160 H160E 0.86 0.52 160 H160F 0.77 0.79 160 H160G 0.82 0.36 160 H160I 0.36 0.58 160 H160L 1.03 0.92 160 H160M 0.56 0.97 160 H160N 1.11 1.02 160 H160Q 0.98 0.47 160 H160R 0.54 0.62 160 H160T 1.01 0.91 160 H160V 0.76 0.74 160 H160W 0.26 0.66 160 H160Y 0.86 0.89 165 D165A 0.53 0.12 165 D165C 1.01 0.07 165 D165E 1.14 0.07 165 D165G 0.63 0.20 165 D165M 0.58 0.10 165 D165N 1.16 1.10 165 D165Q 0.53 0.11 165 D165S 0.83 0.43 168 E168A 0.83 0.92 168 E168C 0.83 0.50 168 E168D 0.82 0.57 168 E168F 0.69 0.59 168 E168G 0.92 0.75 168 E168H 0.84 0.90 168 E168I 1.08 0.71 168 E168L 0.80 0.92 168 E168M 1.12 0.80 168 E168N 0.97 0.83 168 E168Q 0.88 0.87 168 E168R 1.18 0.90 168 E168S 0.95 0.83 168 E168T 0.83 0.16 168 E168V 0.89 0.73 168 E168W 1.23 0.66 168 E168Y 0.76 0.82 172 L172A 1.14 1.06 172 L172C 1.07 0.89 172 L172D 0.83 0.91 172 L172E 0.97 1.01 172 L172G 0.50 0.60 172 L172H 0.93 1.06 172 L172I 0.97 0.90 172 L172K 0.98 1.12 172 L172M 0.86 0.91 172 L172N 0.91 0.96 172 L172P 0.17 0.83 172 L172Q 1.00 0.89 172 L172R 1.16 1.06 172 L172S 0.78 1.01 172 L172T 0.82 0.94 172 L172V 1.02 0.88 172 L172W 1.09 0.92 172 L172Y 1.06 0.98 173 S173A 0.92 0.74 173 S173C 0.82 0.57 173 S173D 0.63 0.71 173 S173E 1.07 0.65 173 S173F 0.82 0.25 173 S173G 0.73 0.78 173 S173H 0.85 0.66 173 S173I 1.20 0.59 173 S173K 1.17 1.03 173 S173L 0.75 0.20 173 S173M 1.05 0.48 173 S173N 1.02 0.84 173 S173Q 1.08 0.84 173 S173R 0.88 1.03 173 S173T 1.33 0.86 173 S173V 1.12 0.46 173 S173W 0.86 0.20 173 S173Y 0.90 0.25 177 K177L 0.89 0.91 177 K177M 0.10 0.90 177 K177Q 0.08 1.07 177 K177R 0.47 1.09 188 E188P 1.40 1.16 191 T191A 0.49 1.11 191 T191C 0.13 1.07 191 T191D 0.91 1.03 191 T191G 0.19 1.09 191 T191I 0.18 1.06 191 T191M 0.06 1.09 191 T191N 0.76 1.13 191 T191P 0.99 1.07 191 T191Q 0.18 1.17 191 T191S 0.72 1.05 191 T191V 0.16 1.02 192 E192C 0.55 1.12 192 E192D 0.42 1.50 192 E192Q 0.22 1.55 192 E192T 0.10 1.35 193 N193C 0.73 0.62 193 N193F 1.71 0.98 193 N193H 1.10 0.92 193 N193K 1.28 1.17 193 N193L 1.22 0.78 193 N193M 0.81 0.96 193 N193R 0.87 0.97 193 N193W 1.09 0.73 193 N193Y 1.89 1.10 196 Y196A 0.74 1.57 196 Y196D 0.29 1.29 196 Y196F 0.74 1.38 196 Y196N 0.54 0.94 196 Y196S 0.36 1.59 199 L199V 0.61 0.13 200 M200A 1.03 0.68 200 M200C 0.84 0.53 200 M200D 0.71 0.81 200 M200E 0.54 0.55 200 M200I 1.14 0.57 200 M200L 0.68 1.11 200 M200N 0.46 0.72 200 M200Q 0.78 0.77 200 M200S 0.61 1.11 200 M200T 0.80 0.61 200 M200V 0.97 0.56 201 Y201A 0.90 1.41 201 Y201C 1.22 0.14 201 Y201D 0.60 0.73 201 Y201E 0.81 1.36 201 Y201F 0.85 0.81 201 Y201G 0.56 1.63 201 Y201H 1.06 1.44 201 Y201I 1.35 0.11 201 Y201K 0.89 0.08 201 Y201L 1.05 0.18 201 Y201M 1.16 1.21 201 Y201N 1.15 0.31 201 Y201Q 1.11 0.79 201 Y201R 0.87 0.06 201 Y201S 0.74 1.11 201 Y201T 0.65 0.39 201 Y201W 0.73 0.08 202 A202C 0.97 0.57 202 A202D 0.83 0.93 202 A202E 0.49 0.85 202 A202G 0.45 0.83 202 A202I 0.50 1.02 202 A202L 0.46 0.95 202 A202M 0.32 0.84 202 A202N 0.53 1.08 202 A202Q 0.47 1.01 202 A202S 0.69 0.79 202 A202T 0.63 1.07 202 A202V 0.82 1.02 213 T213A 1.11 0.98 213 T213C 0.97 0.77 213 T213D 1.12 0.91 213 T213E 1.11 0.88 213 T213F 1.13 0.75 213 T213G 1.11 0.91 213 T213H 0.92 1.00 213 T213K 0.90 1.11 213 T213L 1.26 0.75 213 T213M 1.26 0.78 213 T213N 1.11 0.91 213 T213P 0.94 0.91 213 T213Q 1.12 1.02 213 T213R 1.05 1.05 213 T213S 1.10 1.08 213 T213V 1.35 0.76 213 T213W 1.17 0.68 216 K216A 0.66 0.24 216 K216E 1.03 1.30 216 K216G 0.83 1.20 216 K216H 0.90 1.28 216 K216M 0.97 1.39 216 K216P 0.91 0.97 216 K216Q 1.04 1.34 216 K216R 0.77 1.32 216 K216S 0.97 1.28 216 K216T 0.99 1.22 216 K216V 0.95 1.07 216 K216W 1.00 1.13 216 K216Y 0.79 1.31 217 N217A 1.10 0.87 217 N217C 0.81 0.78 217 N217F 0.90 0.88 217 N217G 0.95 0.90 217 N217H 1.09 0.90 217 N217I 1.08 0.76 217 N217L 1.09 0.82 217 N217M 0.97 0.80 217 N217P 0.97 0.73 217 N217Q 1.31 0.74 217 N217R 1.19 0.87 217 N217S 1.05 0.87 217 N217T 1.01 0.87 217 N217V 1.18 0.69 217 N217W 0.99 0.80 220 K220A 1.06 0.79 220 K220C 1.05 0.75 220 K220D 1.02 0.88 220 K220E 1.12 0.88 220 K220F 1.03 0.78 220 K220G 1.10 0.84 220 K220H 1.12 0.81 220 K220I 1.13 0.81 220 K220M 1.05 0.75 220 K220N 1.17 0.80 220 K220P 1.33 0.89 220 K220Q 1.21 0.87 220 K220R 1.26 0.83 220 K220S 1.30 0.81 220 K220V 1.21 0.82 220 K220W 1.01 0.81 220 K220Y 1.08 0.84 221 W221A 0.88 1.54 221 W221C 0.95 1.09 221 W221D 0.84 1.31 221 W221F 1.07 1.28 221 W221I 1.33 1.23 221 W221L 0.88 1.50 221 W221M 1.16 1.35 221 W221N 1.11 1.57 221 W221R 0.93 1.29 221 W221S 1.34 1.40 221 W221V 1.13 1.31 221 W221Y 1.14 1.36 227 N227A 1.01 1.02 227 N227C 0.92 0.95 227 N227D 1.01 1.06 227 N227E 1.03 1.06 227 N227F 0.72 0.81 227 N227G 1.05 1.09 227 N227H 0.95 1.13 227 N227I 1.03 0.76 227 N227K 1.00 1.13 227 N227L 0.84 0.75 227 N227M 0.84 0.87 227 N227P 1.08 0.88 227 N227Q 0.94 1.00 227 N227R 0.89 1.03 227 N227S 0.96 0.95 227 N227T 1.06 0.96 227 N227V 1.05 0.84 227 N227W 1.07 0.81 227 N227Y 1.01 0.85 232 R232H 0.66 0.34 232 R232K 0.52 0.47 232 R232M 0.62 0.12 232 R232Q 0.54 0.12 232 R232S 0.59 0.16 232 R232T 0.76 0.17 232 R232V 0.70 0.15 235 A235C 0.86 0.53 235 A235D 0.70 0.98 235 A235E 0.93 0.84 235 A235F 1.01 0.68 235 A235G 1.17 0.78 235 A235H 0.80 1.01 235 A235I 1.07 0.84 235 A235K 0.93 1.14 235 A235L 0.89 0.97 235 A235M 0.99 0.91 235 A235N 0.78 1.03 235 A235P 0.97 0.48 235 A235Q 1.01 0.89 235 A235R 1.03 1.14 235 A235S 0.92 1.00 235 A235V 1.01 0.86 235 A235W 0.98 0.60 235 A235Y 0.91 0.93 237 K237R 0.48 0.88 238 H238N 0.21 0.83 240 K240M 0.31 1.13 240 K240Q 0.12 1.21 240 K240R 0.27 1.41 246 D246A 0.73 1.03 246 D246E 1.18 1.03 246 D246F 0.67 1.02 246 D246G 0.61 1.09 246 D246H 0.71 1.05 246 D246I 0.75 0.85 246 D246K 0.36 1.18 246 D246L 0.81 0.91 246 D246M 0.80 0.92 246 D246N 0.68 0.97 246 D246P 0.47 0.81 246 D246Q 0.78 0.98 246 D246R 0.24 1.31 246 D246S 0.97 1.01 246 D246T 0.83 1.14 246 D246Y 0.90 0.96 249 S249A 1.06 0.97 249 S249C 0.93 0.74 249 S249D 0.98 0.94 249 S249E 1.27 0.92 249 S249F 0.91 0.74 249 S249G 0.91 0.94 249 S249H 1.04 0.93 249 S249K 1.15 1.02 249 S249L 1.14 0.82 249 S249M 0.95 0.77 249 S249P 1.09 0.80 249 S249Q 1.20 0.94 249 S249R 1.07 1.03 249 S249T 1.17 0.91 249 S249V 1.01 0.74 249 S249W 1.13 0.77 249 S249Y 1.07 0.87 250 Y250A 0.99 1.21 250 Y250C 1.03 1.12 250 Y250D 0.97 1.29 250 Y250E 1.13 1.33 250 Y250F 1.29 1.28 250 Y250G 1.09 1.33 250 Y250I 1.35 1.27 250 Y250K 1.07 1.48 250 Y250L 1.02 1.32 250 Y250M 1.35 1.39 250 Y250N 1.05 1.40 250 Y250P 0.71 1.05 250 Y250Q 1.01 1.54 250 Y250R 0.99 1.55 250 Y250S 1.02 1.41 250 Y250W 0.99 1.35 252 R252A 1.12 1.08 252 R252C 0.97 0.81 252 R252D 0.89 0.86 252 R252E 1.09 1.12 252 R252F 1.01 0.89 252 R252G 0.76 1.00 252 R252I 1.07 0.97 252 R252K 1.19 1.21 252 R252L 1.32 0.96 252 R252M 0.98 0.96 252 R252N 1.15 0.97 252 R252P 0.72 0.83 252 R252Q 1.16 1.04 252 R252S 1.04 1.01 252 R252T 1.09 0.99 252 R252V 1.01 0.94 252 R252Y 1.14 0.86 253 S253A 1.09 0.97 253 S253D 1.07 1.04 253 S253F 1.19 0.82 253 S253G 1.18 0.92 253 S253H 1.13 0.97 253 S253I 1.13 0.84 253 S253K 1.10 1.01 253 S253L 1.09 0.79 253 S253N 1.06 1.03 253 S253P 0.95 0.90 253 S253Q 1.13 0.93 253 S253T 1.14 0.97 253 S253V 1.15 0.90 253 S253W 1.04 0.87 253 S253Y 1.34 0.94 254 Q254A 0.98 0.88 254 Q254C 0.94 0.66 254 Q254D 1.10 0.90 254 Q254E 1.29 0.89 254 Q254F 1.23 0.74 254 Q254G 1.15 0.77 254 Q254H 1.04 0.94 254 Q254I 1.12 0.91 254 Q254K 1.00 0.99 254 Q254L 1.09 0.82 254 Q254M 0.94 0.89 254 Q254N 1.17 0.90 254 Q254R 1.05 0.98 254 Q254S 1.07 0.98 254 Q254T 1.21 0.65 254 Q254V 1.31 0.92 254 Q254W 1.17 0.69 254 Q254Y 1.03 0.87 255 T255A 1.09 0.73 255 T255C 0.89 0.78 255 T255E 1.09 0.64 255 T255F 1.30 0.68 255 T255G 1.15 0.73 255 T255H 1.10 0.74 255 T255I 1.18 0.70 255 T255K 1.27 0.83 255 T255L 0.97 0.73 255 T255M 0.98 0.72 255 T255N 0.83 0.76 255 T255P 0.77 0.59 255 T255R 1.12 0.85 255 T255S 1.10 0.84 255 T255V 1.17 0.70 255 T255W 1.27 0.74 255 T255Y 1.02 0.72 257 K257A 1.08 0.67 257 K257C 0.89 0.49 257 K257D 1.16 0.75 257 K257E 1.15 0.76 257 K257F 1.03 0.92 257 K257G 0.97 0.73 257 K257H 1.12 0.69 257 K257I 1.09 0.59 257 K257L 1.26 0.74 257 K257M 1.29 0.79 257 K257N 1.16 0.83 257 K257P 0.62 0.38 257 K257Q 1.18 0.82 257 K257R 1.03 0.89 257 K257S 1.29 0.71 257 K257T 1.04 0.77 257 K257V 1.31 0.78 257 K257W 0.99 0.72 258 P258A 0.97 1.08 258 P258C 1.17 0.85 258 P258D 1.33 1.10 258 P258E 0.95 1.05 258 P258F 0.96 0.75 258 P258G 1.30 1.02 258 P258H 1.38 1.13 258 P258I 1.27 0.25 258 P258K 1.29 1.11 258 P258L 1.08 0.61 258 P258M 1.09 0.91 258 P258N 1.07 1.01 258 P258Q 1.31 1.13 258 P258R 1.02 1.13 258 P258S 1.12 1.08 258 P258T 1.27 1.10 258 P258V 1.29 0.80 258 P258W 1.14 0.87 258 P258Y 1.16 1.08 268 Y268A 0.86 1.39 268 Y268C 0.47 1.10 268 Y268D 0.59 1.44 268 Y268E 0.55 1.47 268 Y268F 1.28 1.07 268 Y268G 1.03 1.21 268 Y268H 0.87 1.24 268 Y268K 0.78 1.90 268 Y268L 0.72 1.10 268 Y268M 0.97 1.15 268 Y268N 0.69 1.51 268 Y268P 0.78 1.41 268 Y268Q 0.71 1.30 268 Y268R 0.76 1.49 268 Y268S 1.06 1.22 268 Y268T 0.99 1.12 268 Y268V 0.88 0.99 268 Y268W 0.97 1.07 272 K272R 0.90 0.86 274 H274A 0.66 1.40 274 H274C 0.65 0.68 274 H274D 0.64 1.20 274 H274E 0.86 1.14 274 H274F 0.88 1.00 274 H274G 0.56 1.36 274 H274I 0.76 1.39 274 H274K 0.85 1.60 274 H274L 0.87 1.40 274 H274N 0.67 1.50 274 H274Q 0.84 1.47 274 H274R 0.80 1.50 274 H274S 0.67 1.28 274 H274T 0.69 1.38 274 H274W 1.26 0.79 274 H274Y 1.05 1.07 275 N275A 0.32 1.01 275 N275C 0.22 0.68 275 N275D 0.08 1.03 275 N275G 0.18 1.00 275 N275H 0.60 1.10 275 N275I 0.15 0.87 275 N275K 0.22 1.22 275 N275L 0.20 1.02 275 N275M 0.20 0.96 275 N275Q 0.21 0.96 275 N275R 0.28 1.04 275 N275S 0.28 0.92 275 N275T 0.19 0.79 275 N275V 0.19 0.76 275 N275W 0.05 0.76 275 N275Y 0.89 0.93 283 T283A 1.06 0.97 283 T283C 1.16 0.78 283 T283D 0.92 1.03 283 T283E 0.95 1.01 283 T283G 0.97 1.01 283 T283H 1.09 0.84 283 T283I 1.10 0.72 283 T283K 1.14 1.01 283 T283L 1.07 0.76 283 T283M 1.26 0.93 283 T283N 1.29 0.96 283 T283P 0.46 0.56 283 T283R 0.82 1.08 283 T283S 1.02 1.06 283 T283V 1.23 0.81 283 T283W 1.07 0.75 283 T283Y 1.01 1.04 285 S285A 0.93 0.80 285 S285C 0.73 0.61 285 S285D 0.91 1.09 285 S285E 1.33 0.89 285 S285F 1.18 1.02 285 S285H 0.98 1.10 285 S285I 0.84 0.52 285 S285K 1.16 0.84 285 S285L 0.85 0.54 285 S285M 0.98 0.76 285 S285Q 1.38 1.22 285 S285R 0.84 0.96 285 S285T 0.98 0.79 285 S285V 0.70 0.63 285 S285W 1.13 1.08 285 S285Y 0.97 1.49 293 N293A 1.02 0.93 293 N293C 0.78 0.69 293 N293D 1.08 0.89 293 N293E 0.87 0.92 293 N293F 0.89 0.70 293 N293G 1.31 0.92 293 N293H 1.12 1.05 293 N293I 0.94 0.75 293 N293K 1.42 1.41 293 N293L 0.87 0.81 293 N293M 0.95 1.07 293 N293P 0.97 0.40 293 N293Q 1.14 1.06 293 N293R 0.86 1.37 293 N293S 0.93 0.95 293 N293T 1.10 1.12 293 N293V 1.04 0.82 293 N293W 1.09 0.78 293 N293Y 1.19 0.74 294 K294A 0.83 0.92 294 K294C 0.83 0.50 294 K294D 0.82 0.57 294 K294E 0.83 0.68 294 K294F 0.69 0.59 294 K294G 0.92 0.75 294 K294H 0.84 0.90 294 K294I 1.08 0.71 294 K294L 0.80 0.92 294 K294M 1.12 0.80 294 K294N 0.97 0.83 294 K294Q 0.88 0.87 294 K294R 1.18 0.90 294 K294S 0.95 0.83 294 K294T 0.83 0.16 294 K294V 0.89 0.73 294 K294W 1.23 0.66 294 K294Y 0.76 0.82 297 T297C 0.86 0.53 297 T297D 0.70 0.98 297 T297E 0.93 0.84 297 T297F 1.01 0.68 297 T297G 1.17 0.78 297 T297H 0.80 1.01 297 T297I 1.07 0.84 297 T297K 0.93 1.14 297 T297L 0.89 0.97 297 T297M 0.99 0.91 297 T297N 0.78 1.03 297 T297P 0.97 0.48 297 T297Q 1.01 0.89 297 T297R 1.03 1.14 297 T297S 0.92 1.00 297 T297V 1.01 0.86 297 T297W 0.98 0.60 297 T297Y 0.91 0.93 300 K300A 0.99 0.79 300 K300C 0.95 0.39 300 K300D 0.91 0.61 300 K300E 0.86 0.78 300 K300F 0.74 0.63 300 K300G 0.98 0.62 300 K300H 1.04 0.83 300 K300I 1.02 0.82 300 K300L 0.91 0.73 300 K300M 1.17 0.80 300 K300N 1.02 0.80 300 K300Q 0.90 0.86 300 K300R 1.20 0.92 300 K300S 0.93 0.80 300 K300T 1.16 0.87 300 K300V 1.15 0.84 300 K300W 0.97 0.57 301 S301A 1.10 0.89 301 S301E 1.12 0.94 301 S301F 1.44 0.68 301 S301G 1.02 1.05 301 S301H 1.12 0.87 301 S301I 1.28 0.74 301 S301K 1.08 1.05 301 S301L 1.09 0.97 301 S301M 1.09 0.87 301 S301N 1.16 0.64 301 S301P 1.21 0.61 301 S301Q 1.18 0.95 301 S301R 1.35 0.89 301 S301T 1.23 0.85 301 S301V 1.18 0.81 301 S301W 1.27 0.75 301 S301Y 1.10 0.80 306 D306A 0.82 0.40 306 D306C 0.74 0.30 306 D306E 0.80 0.71 306 D306F 0.71 0.10 306 D306G 0.76 0.26 306 D306H 0.84 0.35 306 D306I 0.80 0.18 306 D306K 0.77 0.41 306 D306L 0.78 0.18 306 D306N 1.15 0.89 306 D306P 0.82 0.39 306 D306Q 1.03 0.43 306 D306R 0.82 0.27 306 D306S 0.81 0.50 306 D306T 0.88 0.29 306 D306V 0.99 0.22 306 D306Y 0.94 0.12 309 T309C 1.15 0.59 309 T309D 1.27 0.89 309 T309E 0.95 0.91 309 T309F 1.15 0.80 309 T309G 1.17 1.00 309 T309H 0.94 0.97 309 T309I 1.17 0.82 309 T309K 1.18 1.08 309 T309L 1.15 0.95 309 T309M 1.15 0.97 309 T309N 1.20 0.99 309 T309P 0.93 0.20 309 T309Q 1.19 0.98 309 T309R 1.12 1.08 309 T309S 1.00 1.04 309 T309V 1.38 0.95 309 T309W 1.08 0.77 309 T309Y 1.11 0.94 312 T312A 1.01 1.00 312 T312C 0.99 0.70 312 T312D 1.03 0.96 312 T312E 1.15 0.95 312 T312F 1.05 0.92 312 T312G 1.18 1.07 312 T312H 1.30 0.99 312 T312K 0.83 0.25 312 T312L 1.08 0.95 312 T312M 0.98 0.91 312 T312N 1.04 0.99 312 T312P 0.74 0.85 312 T312Q 1.05 0.94 312 T312R 1.13 1.00 312 T312S 1.29 0.99 312 T312V 1.40 0.87 312 T312W 1.14 0.83 312 T312Y 1.31 0.92 313 N313A 1.01 0.93 313 N313C 0.95 0.63 313 N313D 0.95 0.51 313 N313E 1.05 0.90 313 N313F 1.06 0.64 313 N313G 1.25 0.96 313 N313H 1.25 0.94 313 N313I 1.44 0.55 313 N313K 1.12 0.85 313 N313L 1.21 0.85 313 N313M 1.02 0.89 313 N313P 1.05 0.81 313 N313Q 1.00 1.00 313 N313R 1.19 1.13 313 N313S 1.25 1.05 313 N313V 1.28 0.74 313 N313W 1.01 0.67 313 N313Y 1.10 0.90 317 K317A 0.98 0.94 317 K317C 0.83 0.54 317 K317D 0.82 0.86 317 K317E 0.78 0.91 317 K317F 0.92 0.84 317 K317G 0.91 0.88 317 K317L 1.10 0.86 317 K317M 1.02 0.95 317 K317N 1.03 0.92 317 K317P 0.86 0.80 317 K317Q 0.76 0.94 317 K317R 0.78 0.89 317 K317S 1.04 0.93 317 K317T 0.94 0.88 317 K317V 1.00 0.93 317 K317W 1.08 0.83 317 K317Y 1.05 0.93 318 D318A 0.93 1.38 318 D318E 0.90 1.09 318 D318F 0.78 1.22 318 D318G 0.91 1.39 318 D318H 1.12 1.10 318 D318I 0.75 1.40 318 D318K 0.65 1.73 318 D318L 1.00 1.31 318 D318M 0.90 1.26 318 D318N 0.92 1.19 318 D318P 0.61 0.37 318 D318Q 0.93 1.14 318 D318R 0.71 1.54 318 D318S 1.11 1.37 318 D318T 1.40 1.32 318 D318V 0.81 1.34 318 D318W 0.90 1.07 318 D318Y 1.10 1.33 319 Q319A 1.02 1.13 319 Q319C 0.73 1.38 319 Q319D 0.85 1.31 319 Q319E 0.98 1.20 319 Q319F 0.87 1.11 319 Q319G 1.14 1.03 319 Q319H 0.94 1.28 319 Q319I 0.94 1.32 319 Q319K 1.10 1.52 319 Q319L 0.95 1.11 319 Q319M 0.90 1.09 319 Q319N 0.91 1.12 319 Q319P 1.13 0.57 319 Q319R 1.18 1.44 319 Q319S 0.91 1.12 319 Q319T 0.98 1.10 319 Q319V 1.07 1.08 319 Q319W 1.05 1.08 319 Q319Y 1.04 1.41 320 P320A 1.02 0.96 320 P320C 1.01 0.75 320 P320D 0.74 0.91 320 P320E 1.04 0.85 320 P320F 0.76 0.77 320 P320G 1.00 1.00 320 P320H 1.00 1.18 320 P320I 0.86 0.80 320 P320K 0.96 1.23 320 P320L 0.87 0.83 320 P320M 1.04 0.60 320 P320Q 0.95 1.08 320 P320R 0.79 1.25 320 P320S 1.16 1.03 320 P320T 1.11 1.28 320 P320V 1.08 0.88 320 P320W 0.90 1.03 320 P320Y 1.05 1.03 338 L338A 1.36 1.29 338 L338C 1.24 0.67 338 L338D 1.00 0.94 338 L338E 0.87 0.65 338 L338F 0.90 0.17 338 L338G 1.38 1.34 338 L338H 0.05 0.05 338 L338I 1.12 1.32 338 L338M 1.20 1.27 338 L338P 1.11 1.23 338 L338Q 0.96 0.61 338 L338S 1.13 1.51 338 L338T 1.42 1.05 338 L338V 1.14 1.55 338 L338W 0.98 0.14 338 L338Y 1.15 0.11 339 Q339A 1.08 1.13 339 Q339C 0.88 0.79 339 Q339D 0.93 0.11 339 Q339E 1.07 0.84 339 Q339F 0.86 0.55 339 Q339G 1.17 1.21 339 Q339H 1.03 1.04 339 Q339K 1.26 1.13 339 Q339L 1.12 0.70 339 Q339M 0.93 0.81 339 Q339P 1.02 1.24 339 Q339R 0.81 0.35 339 Q339S 1.02 1.02 339 Q339T 1.35 1.01 339 Q339V 1.23 0.76 339 Q339Y 1.14 0.78 340 S340A 1.23 1.43 340 S340C 0.74 0.75 340 S340D 0.97 1.63 340 S340E 0.92 1.58 340 S340F 0.83 0.82 340 S340H 1.12 1.45 340 S340I 1.07 1.07 340 S340K 0.99 1.76 340 S340M 1.24 1.20 340 S340N 1.10 1.75 340 S340P 0.69 0.81 340 S340Q 1.21 1.76 340 S340T 1.21 1.14 340 S340V 1.00 1.09 340 S340Y 1.02 0.97 343 D343A 0.96 0.35 343 D343C 1.32 0.74 343 D343E 1.00 1.07 343 D343F 0.91 0.79 343 D343H 0.98 1.02 343 D343I 1.27 0.88 343 D343L 0.95 1.08 343 D343M 0.99 1.02 343 D343N 1.05 0.88 343 D343P 1.30 1.03 343 D343Q 1.14 1.01 343 D343R 1.25 1.03 343 D343T 1.08 0.98 343 D343W 1.00 0.64 343 D343Y 1.29 0.82 345 W345A 1.05 0.90 345 W345C 0.97 0.43 345 W345D 1.10 1.15 345 W345E 1.06 1.24 345 W345F 1.07 0.55 345 W345H 1.15 1.10 345 W345I 1.28 0.90 345 W345L 1.07 0.99 345 W345M 1.02 1.01 345 W345N 1.07 1.10 345 W345P 1.00 0.94 345 W345Q 1.26 1.10 345 W345S 1.01 1.12 345 W345T 1.15 1.15 345 W345V 1.16 1.02 363 C363A 0.84 1.06 363 C363D 0.87 1.74 363 C363E 0.99 1.34 363 C363F 0.83 1.03 363 C363G 0.61 0.83 363 C363H 0.78 0.76 363 C363I 0.92 0.63 363 C363L 0.73 0.89 363 C363M 0.97 1.36 363 C363N 0.92 1.86 363 C363Q 0.88 1.78 363 C363S 0.88 1.35 363 C363T 1.15 0.18 363 C363V 1.02 0.99 363 C363W 0.35 0.70 363 C363Y 0.92 0.12 366 Y366A 0.96 1.14 366 Y366D 0.52 1.18 366 Y366E 0.91 1.18 366 Y366F 0.91 0.87 366 Y366G 0.94 1.08 366 Y366H 1.07 1.12 366 Y366I 0.85 0.87 366 Y366K 0.72 0.82 366 Y366L 0.77 0.61 366 Y366M 0.92 0.79 366 Y366N 1.03 0.91 366 Y366P 0.54 0.78 366 Y366Q 1.03 1.49 366 Y366R 0.96 0.96 366 Y366S 1.07 1.02 366 Y366T 1.01 0.91 366 Y366V 1.04 0.94 366 Y366W 1.11 0.99 369 Y369E 0.98 0.87 369 Y369F 1.03 0.79 369 Y369G 0.86 0.33 369 Y369H 0.89 0.78 369 Y369I 1.33 0.91 369 Y369K 1.07 0.80 369 Y369M 1.06 1.02 369 Y369P 0.49 0.20 369 Y369Q 1.07 0.79 369 Y369R 1.11 0.95 369 Y369S 0.89 0.60 369 Y369T 1.28 0.68 369 Y369V 1.17 0.91 369 Y369W 1.09 0.95 370 Y370A 1.03 1.21 370 Y370D 0.48 1.35 370 Y370E 0.98 1.35 370 Y370F 0.90 0.73 370 Y370G 1.21 1.18 370 Y370H 0.96 1.36 370 Y370I 0.99 1.00 370 Y370K 0.93 1.65 370 Y370L 0.93 0.88 370 Y370M 0.91 1.04 370 Y370N 1.04 1.41 370 Y370P 0.44 0.67 370 Y370Q 0.87 1.51 370 Y370S 1.06 1.50 370 Y370T 1.07 1.10 370 Y370V 1.05 1.13 370 Y370W 0.94 0.91 375 Y375A 1.03 1.39 375 Y375C 0.59 0.48 375 Y375D 0.96 1.52 375 Y375E 0.96 1.48 375 Y375F 0.90 1.00 375 Y375G 0.90 0.98 375 Y375H 0.98 1.16 375 Y375I 0.94 1.06 375 Y375K 0.96 1.43 375 Y375L 1.03 1.07 375 Y375M 0.98 1.05 375 Y375N 0.92 1.48 375 Y375P 0.92 0.89 375 Y375Q 0.92 1.56 375 Y375R 0.77 1.61 375 Y375S 0.92 1.29 375 Y375T 1.25 1.04 375 Y375W 0.98 0.88 379 S379A 1.01 1.02 379 S379C 0.60 0.44 379 S379D 0.92 0.96 379 S379E 0.99 1.01 379 S379G 0.90 0.91 379 S379I 0.80 0.70 379 S379K 1.00 1.12 379 S379L 0.84 0.56 379 S379M 0.87 0.80 379 S379N 1.03 0.98 379 S379P 0.61 0.39 379 S379Q 0.94 0.98 379 S379R 0.96 1.01 379 S379T 1.07 0.95 379 S379V 0.90 0.75 379 S379W 0.70 0.35 379 S379Y 0.92 0.59 381 K381A 0.85 0.78 381 K381C 0.86 0.35 381 K381D 0.87 0.65 381 K381E 0.93 0.81 381 K381F 0.96 0.20 381 K381G 0.96 0.82 381 K381H 1.13 0.73 381 K381I 0.98 0.36 381 K381L 0.95 0.38 381 K381M 0.93 0.56 381 K381N 0.87 0.68 381 K381P 1.18 0.39 381 K381Q 1.03 0.90 381 K381R 1.20 0.95 381 K381S 1.18 0.89 381 K381T 1.01 0.60 381 K381V 1.00 0.43 381 K381W 0.90 0.22 381 K381Y 0.87 0.63 385 D385A 1.01 0.88 385 D385E 0.89 1.05 385 D385F 0.73 0.54 385 D385G 1.05 0.88 385 D385H 0.96 0.99 385 D385K 1.00 1.06 385 D385L 0.96 0.47 385 D385N 0.91 0.96 385 D385Q 1.02 1.01 385 D385R 0.86 0.95 385 D385S 1.10 1.00 385 D385T 1.22 0.92 385 D385V 0.85 0.43 385 D385W 0.98 0.53 386 P386A 0.90 0.80 386 P386C 0.72 0.69 386 P386D 0.85 0.94 386 P386E 0.94 0.87 386 P386F 0.72 0.66 386 P386G 1.02 0.77 386 P386H 0.89 0.93 386 P386I 1.12 0.73 386 P386K 1.22 0.87 386 P386L 0.96 0.73 386 P386M 0.94 0.70 386 P386N 0.91 0.86 386 P386Q 0.95 0.86 386 P386S 0.83 0.82 386 P386T 1.00 0.54 386 P386V 1.11 0.79 386 P386W 0.90 0.44 386 P386Y 0.91 0.78 391 R391A 0.58 0.22 391 R391H 0.59 0.29 391 R391K 0.88 0.59 391 R391N 0.71 0.38 391 R391Q 0.62 0.28 391 R391T 0.67 0.25 392 R392A 0.89 0.73 392 R392C 0.74 0.66 392 R392E 0.79 0.46 392 R392F 1.03 0.43 392 R392G 0.99 0.65 392 R392H 0.86 0.96 392 R392I 1.08 0.57 392 R392K 1.10 1.09 392 R392L 0.91 0.63 392 R392M 1.07 0.72 392 R392N 0.89 0.90 392 R392P 0.67 0.31 392 R392Q 1.12 0.75 392 R392S 1.00 0.73 392 R392T 1.00 0.91 392 R392V 0.89 0.48 392 R392W 0.68 0.23 392 R392Y 1.00 0.60 393 D393A 0.98 0.77 393 D393C 0.69 0.48 393 D393E 0.92 0.81 393 D393F 0.84 0.61 393 D393G 1.08 0.75 393 D393H 0.88 0.75 393 D393K 1.09 0.80 393 D393L 1.04 0.70 393 D393Q 1.00 0.82 393 D393R 0.88 0.64 393 D393S 0.92 0.91 393 D393T 1.12 0.90 393 D393V 1.04 0.63 393 D393W 0.95 0.66 393 D393Y 1.01 0.66 394 Y394A 0.91 0.86 394 Y394D 0.98 0.84 394 Y394E 0.92 1.03 394 Y394F 1.07 0.98 394 Y394G 1.13 0.85 394 Y394H 1.04 0.99 394 Y394I 1.11 0.95 394 Y394K 1.09 1.07 394 Y394L 1.22 1.11 394 Y394M 0.74 0.23 394 Y394N 1.00 1.01 394 Y394Q 1.09 1.13 394 Y394S 1.11 1.15 394 Y394V 3.00 0.75 394 Y394W 1.11 1.16 400 H400A 1.24 0.89 400 H400C 1.16 0.73 400 H400D 1.05 0.82 400 H400E 0.99 0.95 400 H400F 1.01 0.94 400 H400G 0.90 0.83 400 H400I 1.04 0.91 400 H400K 0.92 1.03 400 H400L 0.90 0.88 400 H400M 1.01 0.91 400 H400N 1.26 0.92 400 H400Q 0.96 0.94 400 H400R 1.03 0.87 400 H400S 0.94 0.92 400 H400T 0.95 0.88 400 H400V 1.28 0.91 400 H400W 1.17 0.80 400 H400Y 1.15 0.92 402 Y402A 1.07 0.97 402 Y402C 0.92 0.76 402 Y402D 0.90 0.80 402 Y402E 1.09 0.77 402 Y402F 0.89 0.82 402 Y402G 0.92 0.81 402 Y402H 1.21 0.91 402 Y402I 1.36 0.75 402 Y402K 0.95 0.84 402 Y402L 1.09 0.49 402 Y402M 1.14 0.88 402 Y402N 1.06 0.86 402 Y402P 1.03 0.28 402 Y402Q 0.98 0.83 402 Y402R 1.16 0.75 402 Y402T 1.32 1.02 402 Y402V 1.40 0.95 402 Y402W 1.24 0.89 403 L403A 1.20 0.89 403 L403C 1.10 0.98 403 L403D 1.03 0.95 403 L403E 1.26 0.93 403 L403F 1.03 0.74 403 L403G 1.22 0.96 403 L403H 1.10 0.90 403 L403M 1.11 0.99 403 L403N 0.98 0.95 403 L403P 0.78 0.47 403 L403Q 1.24 0.98 403 L403R 1.36 1.01 403 L403S 1.17 1.00 403 L403T 1.53 0.99 403 L403V 1.34 1.00 403 L403W 1.15 0.85 403 L403Y 1.16 0.97 404 D404A 1.12 0.73 404 D404C 1.28 0.61 404 D404E 1.38 0.78 404 D404G 1.25 0.77 404 D404I 1.20 0.84 404 D404K 1.10 0.83 404 D404L 1.09 0.91 404 D404M 1.13 0.76 404 D404N 1.13 0.98 404 D404P 1.05 0.56 404 D404Q 1.17 0.91 404 D404R 1.15 0.77 404 D404S 1.19 0.99 404 D404V 1.28 0.79 404 D404W 1.05 0.76 404 D404Y 1.08 0.81 406 S406A 0.99 0.99 406 S406C 1.11 0.85 406 S406D 0.93 1.02 406 S406E 0.95 0.91 406 S406F 0.86 0.88 406 S406G 0.93 0.86 406 S406H 0.88 0.98 406 S406I 0.92 0.91 406 S406K 0.95 0.82 406 S406L 0.94 0.98 406 S406M 0.89 0.90 406 S406N 1.09 0.94 406 S406P 0.91 0.93 406 S406T 1.18 0.97 406 S406V 1.14 0.87 406 S406Y 0.99 0.80 407 D407C 1.14 0.41 407 D407E 0.82 0.59 407 D407F 0.88 0.35 407 D407G 1.10 0.38 407 D407H 0.85 0.63 407 D407I 1.05 0.22 407 D407K 1.00 0.44 407 D407L 0.91 0.18 407 D407M 1.05 0.37 407 D407N 1.11 0.96 407 D407Q 0.94 0.53 407 D407R 0.78 0.36 407 D407S 0.93 0.65 407 D407T 1.06 0.49 407 D407V 0.93 0.29 407 D407W 1.06 0.20 407 D407Y 0.85 0.38 410 G410A 0.90 1.00 410 G410C 1.04 0.81 410 G410F 0.96 0.22 410 G410H 0.93 0.34 410 G410M 1.13 0.35 410 G410N 0.99 0.27 410 G410Q 1.05 0.14 410 G410R 0.98 0.27 410 G410T 1.08 0.70 410 G410V 1.10 0.42 410 G410Y 0.92 0.49 413 R413A 1.02 1.06 413 R413D 0.71 0.40 413 R413E 0.86 0.67 413 R413G 1.19 0.33 413 R413H 1.06 0.95 413 R413I 0.96 0.75 413 R413K 1.08 0.95 413 R413L 1.02 0.96 413 R413M 0.81 0.81 413 R413N 0.93 0.72 413 R413Q 0.81 0.35 413 R413S 0.85 0.87 413 R413V 0.93 0.73 413 R413W 0.92 0.41 413 R413Y 0.73 0.49 414 E414A 1.06 0.70 414 E414C 1.05 0.55 414 E414D 1.13 0.75 414 E414F 0.81 0.59 414 E414G 0.82 0.68 414 E414H 0.89 0.65 414 E414I 0.98 0.60 414 E414K 0.96 0.65 414 E414L 1.16 0.71 414 E414M 0.88 0.72 414 E414N 0.99 0.57 414 E414P 0.85 0.60 414 E414Q 0.85 0.70 414 E414R 1.00 0.65 414 E414S 0.91 0.63 414 E414T 0.79 0.67 414 E414W 1.03 0.25 414 E414Y 0.78 0.58 416 V416A 0.93 0.67 416 V416C 0.94 0.61 416 V416D 1.05 0.71 416 V416H 0.92 0.78 416 V416I 0.83 0.74 416 V416K 0.71 0.65 416 V416L 0.96 0.81 416 V416M 1.06 0.78 416 V416N 0.92 0.66 416 V416P 1.18 0.53 416 V416Q 1.02 0.74 416 V416R 1.02 0.29 416 V416S 1.15 0.46 416 V416T 1.01 0.65 416 V416W 0.83 0.55 416 V416Y 0.89 0.69 419 K419A 1.36 1.29 419 K419C 1.24 0.67 419 K419D 1.00 0.94 419 K419E 0.87 0.65 419 K419F 0.90 0.17 419 K419H 0.05 0.05 419 K419I 1.12 1.32 419 K419M 1.20 1.27 419 K419P 1.11 1.23 419 K419Q 0.96 0.61 419 K419S 1.13 1.51 419 K419T 1.42 1.05 419 K419V 1.14 1.55 419 K419W 0.98 0.14 419 K419Y 1.15 0.11 422 S422A 0.64 0.97 422 S422C 0.96 0.71 422 S422D 0.97 0.96 422 S422E 1.31 0.78 422 S422F 0.96 0.71 422 S422G 1.20 0.99 422 S422H 1.06 0.66 422 S422I 1.11 0.85 422 S422K 1.16 0.96 422 S422L 0.99 0.74 422 S422M 1.04 0.94 422 S422N 1.12 1.03 422 S422P 0.84 0.70 422 S422Q 0.15 0.82 422 S422R 1.02 0.94 422 S422T 0.97 0.92 422 S422V 1.17 0.88 422 S422W 0.96 0.70 422 S422Y 1.09 0.92 427 L427A 0.93 0.66 427 L427C 1.02 0.68 427 L427E 0.86 0.27 427 L427F 0.89 0.30 427 L427G 0.63 0.26 427 L427I 1.08 0.64 427 L427M 0.86 0.79 427 L427N 0.76 0.31 427 L427P 1.13 0.06 427 L427Q 0.95 0.53 427 L427S 0.78 0.27 427 L427T 0.80 0.70 427 L427V 0.82 0.72 433 G433A 1.27 1.08 433 G433C 1.15 0.69 433 G433D 1.05 0.96 433 G433E 0.92 0.99 433 G433F 1.04 0.92 433 G433H 1.27 0.99 433 G433I 1.37 0.86 433 G433K 1.27 1.05 433 G433L 1.30 0.90 433 G433M 1.23 1.01 433 G433N 1.07 0.75 433 G433P 1.13 0.95 433 G433Q 0.78 0.99 433 G433R 1.00 0.91 433 G433S 1.17 0.96 433 G433T 1.17 0.90 433 G433V 1.27 0.95 433 G433Y 1.26 1.01 436 K436A 0.92 0.94 436 K436C 0.90 0.84 436 K436D 0.86 0.93 436 K436E 0.70 0.87 436 K436F 0.81 0.64 436 K436G 0.84 0.77 436 K436H 1.09 0.89 436 K436I 1.08 0.81 436 K436L 1.01 0.78 436 K436M 0.76 0.85 436 K436N 0.98 0.92 436 K436P 0.88 0.71 436 K436Q 1.01 0.96 436 K436R 1.06 0.79 436 K436S 0.75 0.92 436 K436T 0.95 0.90 436 K436V 0.98 0.87 436 K436W 1.07 0.71 436 K436Y 0.99 0.80 439 Y439A 1.02 0.78 439 Y439D 1.01 0.85 439 Y439F 0.77 0.78 439 Y439G 1.01 0.77 439 Y439H 0.96 0.73 439 Y439K 0.96 0.74 439 Y439M 1.04 0.77 439 Y439N 0.96 0.83 439 Y439P 0.87 0.85 439 Y439Q 0.90 0.88 439 Y439R 0.75 0.80 439 Y439S 0.94 0.82 439 Y439T 0.84 0.79 439 Y439V 1.04 0.70 439 Y439W 0.86 0.72 442 K442A 1.38 0.98 442 K442F 1.04 0.97 442 K442G 1.23 1.02 442 K442H 1.07 1.04 442 K442I 1.13 0.93 442 K442N 1.39 1.03 442 K442P 1.11 1.03 442 K442Q 1.11 1.05 442 K442R 1.33 1.01 442 K442S 1.24 1.07 442 K442T 1.34 1.06 442 K442V 1.20 0.99 442 K442W 1.32 0.98 442 K442Y 1.24 1.08 445 A445C 0.98 0.83 445 A445D 1.04 0.87 445 A445G 1.21 1.01 445 A445H 0.90 0.93 445 A445I 1.25 0.84 445 A445K 1.20 0.11 445 A445L 1.17 0.92 445 A445N 1.20 0.91 445 A445P 0.91 0.77 445 A445R 0.91 0.89 445 A445S 1.16 0.94 445 A445T 1.29 0.88 445 A445V 1.27 0.93 445 A445W 1.25 0.80 447 K447A 1.09 1.06 447 K447C 1.11 0.87 447 K447D 1.00 0.99 447 K447F 1.09 0.84 447 K447G 1.06 0.94 447 K447H 1.13 0.92 447 K447I 1.22 0.91 447 K447L 1.06 1.01 447 K447M 1.07 0.96 447 K447N 1.43 0.97 447 K447Q 1.34 1.00 447 K447R 1.10 0.96 447 K447S 0.90 0.92 447 K447T 1.21 0.37 447 K447V 0.69 0.86 447 K447W 1.31 0.89 447 K447Y 1.21 0.96 448 V448A 0.98 0.96 448 V448C 1.36 0.98 448 V448D 1.15 1.02 448 V448F 1.48 1.01 448 V448G 1.26 1.05 448 V448H 1.37 1.03 448 V448I 1.44 0.97 448 V448K 1.20 1.07 448 V448L 1.04 1.08 448 V448M 1.13 0.97 448 V448N 1.24 0.70 448 V448P 0.84 1.19 448 V448Q 1.18 1.16 448 V448S 1.20 1.10 448 V448W 1.08 0.89 448 V448Y 1.33 1.27 450 Y450A 0.95 0.94 450 Y450C 1.22 0.84 450 Y450D 1.19 0.95 450 Y450E 1.01 0.92 450 Y450G 1.02 0.93 450 Y450H 1.23 0.90 450 Y450K 1.18 0.94 450 Y450L 0.93 0.69 450 Y450M 1.29 0.89 450 Y450N 1.23 0.96 450 Y450P 0.75 0.30 450 Y450Q 1.00 0.95 450 Y450R 1.22 1.02 450 Y450S 1.22 1.01 450 Y450T 1.32 0.96 450 Y450W 1.21 0.95 452 L452A 1.08 1.06 452 L452C 1.00 1.01 452 L452D 0.98 1.08 452 L452E 0.75 0.55 452 L452F 0.79 0.93 452 L452G 1.07 1.00 452 L452H 1.05 0.99 452 L452K 1.11 1.08 452 L452M 1.13 1.09 452 L452N 1.06 1.28 452 L452P 1.02 0.78 452 L452Q 0.92 1.22 452 L452R 0.93 1.26 452 L452S 0.86 1.21 452 L452T 1.02 1.18 452 L452V 1.14 1.14 452 L452Y 1.17 1.07 455 N455A 1.07 1.04 455 N455C 0.85 0.89 455 N455D 1.07 0.97 455 N455E 1.14 0.94 455 N455G 1.23 1.00 455 N455H 1.05 1.01 455 N455I 1.23 0.95 455 N455K 1.10 1.08 455 N455L 1.06 0.97 455 N455M 0.95 0.96 455 N455P 1.36 0.93 455 N455Q 0.96 0.91 455 N455R 1.13 1.02 455 N455S 1.04 0.91 455 N455T 1.16 0.90 455 N455V 1.26 0.89 455 N455W 1.12 0.76 455 N455Y 1.08 0.15 463 N463A 1.25 1.06 463 N463D 0.97 1.02 463 N463F 1.04 0.87 463 N463G 1.04 1.00 463 N463H 1.12 0.99 463 N463K 1.07 1.00 463 N463L 1.16 1.01 463 N463M 1.24 1.08 463 N463P 0.93 1.05 463 N463Q 0.98 1.04 463 N463R 0.95 0.93 463 N463S 1.27 0.96 463 N463T 1.38 0.91 463 N463V 1.32 0.86 463 N463W 1.45 0.74 463 N463Y 1.20 0.90 465 D465A 0.76 1.06 465 D465C 0.84 0.74 465 D465E 0.95 0.93 465 D465F 0.78 0.89 465 D465G 1.35 0.92 465 D465H 1.06 0.92 465 D465I 1.37 0.85 465 D465K 1.53 0.88 465 D465L 1.14 0.95 465 D465M 1.06 0.98 465 D465N 1.32 0.93 465 D465P 1.13 0.71 465 D465Q 0.86 0.94 465 D465R 1.18 0.90 465 D465S 0.87 0.98 465 D465T 1.42 0.92 465 D465V 1.24 0.93 465 D465W 1.00 0.83 465 D465Y 1.06 0.93 469 E469A 1.16 1.01 469 E469C 1.03 0.86 469 E469D 1.22 1.02 469 E469F 1.11 1.00 469 E469G 1.19 1.00 469 E469H 1.04 0.96 469 E469K 1.16 0.96 469 E469L 1.10 0.98 469 E469N 1.19 0.47 469 E469P 0.85 1.05 469 E469Q 1.03 1.04 469 E469R 1.01 0.75 469 E469S 0.91 1.08 469 E469T 1.15 1.06 469 E469V 1.15 1.08 469 E469W 1.24 0.97 469 E469Y 1.35 1.09 471 K471A 1.09 1.09 471 K471C 1.04 0.91 471 K471D 1.01 1.06 471 K471F 1.10 1.05 471 K471G 1.13 1.12 471 K471H 1.00 1.10 471 K471I 1.22 1.02 471 K471L 0.99 1.07 471 K471M 0.95 1.14 471 K471N 1.04 1.12 471 K471P 0.84 0.98 471 K471Q 0.90 1.08 471 K471R 0.77 1.33 471 K471S 0.97 1.01 471 K471T 1.11 1.09 471 K471V 1.28 1.11 471 K471Y 1.15 1.36 473 N473A 1.03 0.99 473 N473C 1.15 0.74 473 N473D 1.14 0.98 473 N473E 1.20 0.99 473 N473F 1.10 0.83 473 N473G 1.35 0.99 473 N473H 1.02 0.91 473 N473I 0.66 0.45 473 N473K 1.02 1.02 473 N473M 1.11 1.00 473 N473P 1.01 0.95 473 N473Q 1.13 0.99 473 N473R 1.08 1.05 473 N473S 1.15 0.98 473 N473T 1.04 1.04 473 N473W 0.85 0.64 473 N473Y 1.23 0.86 476 S476A 1.51 1.02 476 S476C 0.91 0.89 476 S476D 0.98 0.91 476 S476E 1.08 0.91 476 S476F 1.09 0.87 476 S476G 1.22 0.97 476 S476H 1.07 0.96 476 S476I 1.03 0.78 476 S476K 1.01 0.97 476 S476L 1.46 0.93 476 S476M 1.58 1.08 476 S476N 1.61 0.98 476 S476P 1.02 0.62 476 S476Q 1.13 1.03 476 S476R 1.01 1.08 476 S476T 1.78 1.01 476 S476V 1.21 0.89 476 S476W 1.43 0.78 476 S476Y 1.79 0.94

Example 27 Restrictive Versus Non-Restrictive Positions

Based on the relative performance and stability data for the AmyS positions described in Example 26, AmyS positions were classified as “restrictive” versus “non-restrictive” as follows: Non-restrictive positions have≧20% neutral mutations for at least one property; and restrictive positions have<20% neutral mutations for activity and stability. Non-restrictive positions are good candidates for mutation to design α-amylases having improved function because a large number of mutations are either tolerated (to maintain near wild-type performance) or improved performance. Restrictive positions are not good candidates for mutation because mutations are generally not tolerated. The properties of any amylase can be improved by combining mutations at non-restrictive positions. Table 27-1 shows the two restrictive positions identified in AmyS (%=percent of variants evaluated that meet definition of neutral mutation). Table 27-2 shows the 150 non-restrictive positions identified in AmyS (%=percent of variants evaluated that meet definition of neutral mutation; >20% neutral mutations for at least one property). Restrictive and non-restrictive positions are expected to be conserved among different α-amylases.

TABLE 27-1 Restrictive positions in AmyS Wild type Stability PI Activity PI Position amino acid % >0.5 % >0.5 106 H 18% 0% 199 L 13% 0%

TABLE 27-2 Non-Restrictive positions in AmyS Wild type Satbility PI Activity PI Position amino acid % >0.5 % >0.5 5 N 100%  6% 6 G 100%  100% 13 E 11% 89% 14 W 100%  84% 15 Y 100%  100% 16 L 94% 100% 18 D 100%  100% 20 G 95% 16% 25 K 100%  100% 27 A 100%  89% 29 E 100%  53% 36 L 100%  74% 39 T 100%  80% 50 T 95% 74% 52 R 94% 94% 53 S 100%  100% 54 D 11% 79% 67 E 94% 81% 71 K 95% 100% 73 T 83% 100% 75 R 100%  100% 77 K 24% 94% 80 T 100%  100% 81 K 100%  94% 83 Q 100%  94% 85 L 100%  100% 90 A 100%  95% 92 H 94% 56% 107 K 21% 95% 111 D 74% 95% 113 T 100%  100% 114 E 50% 94% 120 E 17% 100% 121 V  6% 94% 126 R  6% 88% 128 Q 29% 88% 131 S 13% 94% 133 T 53% 100% 137 Q 94% 94% 138 A 39% 72% 139 W 100%  88% 141 K 100%  100% 143 D 93% 80% 147 R 95% 0% 149 N 100%  93% 150 T 100%  88% 151 Y 100%  100% 152 S 94% 47% 155 K 100%  100% 160 H 89% 78% 165 D 47% 11% 168 E 89% 84% 172 L 94% 100% 173 S 100%  67% 177 K  6% 100% 188 E  6% 100% 191 T 24% 100% 192 E  6% 88% 193 N 53% 100% 196 Y 18% 100%

Example 28 Viscosity Reduction by AmyS Variants

Viscosity reduction of different batches of corn flour (bag A, C, E, G) by AmyS variants was monitored as described in Example 6 and was compared to viscosity reduction by SPEZYME® Xtra (Genencor). Results are shown in FIG. 32A and Table 28-1. Improved AmyS variants in the viscometer assay can be identified by a number of criteria: decreased peak viscosity, decreased final viscosity, or a decreased enzyme dose required to produce similar peak or final viscosities relative to the dose required for the wildtype enzyme. In Table 28-1, improved properties of AmyS variants are shown in bold type.

TABLE 28-1 Viscosity reduction of corn flour by AmyS variants corn pared to Xtra dose peak final (ug) viscosity viscosity corn flour bag A Xtra 30.0 20610 4850 (UFC) I181A 27.5 16930 13140 I181P 27.5 17320 13910 corn flour bag C Xtra 30.0 10870 3033 I181C 30.0 11810 1280 I181E 30.0 10800 1200 I181Y 30.0 8990 2495 S242A 30.0 10770 740 S242Q 30.0 8220 440 corn flour bag E Xtra 15.0 18890 2830 S242A 15.0 17300 1165 S242E 15.0 18640 1345 S242Q 15.0 20490 1800 corn flour bag G Xtra 20.0 26300 13100 S242Q 20.0 20433 3660 G132A 20.0 18400 10800 N193Y 20.0 28900 11500 E188P 20.0 24000 6950

Example 29 Viscosity Reduction by AmyS Variants in Presence of Phytase

Viscosity reduction of corn fluor by AmyS N193Y was monitored with and without the addition of Phytase BP111 as described in Example 6 with the following modifications. The effect of viscosity reduction was measured at pH 5.2, pH 5.5 and 5.8. Results are shown in FIGS. 32B and C. Addition of Phytase (BP111) to AmyS N193Y makes significant improvements to the variant's ability to reduce viscosity in the Viscometer.

All publications and patents mentioned above are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the following claims. 

1.-28. (canceled)
 29. A method for hydrolyzing a soluble starch substrate using an α-amylase variant, comprising contacting the starch substrate with an α-amylase variant having alpha amylase activity and at least one altered characteristic that improves enzyme performance, the variant comprising: an amino acid sequence having at least 90% sequence identity to the parental α-amylase polypeptide of SEQ ID NO: 2, and having a substitution that introduces the amino acid residue E188P, wherein the α-amylase variant exhibits increased viscosity reduction in a starch liquefaction assay compared to the parental polypeptide.
 30. The method of claim 29, further comprising contacting the starch substrate with a phytase.
 31. The method of claim 30, wherein the contacting with a phytase is prior to or simultaneous with the contacting with the α-amylase variant.
 32. A method for liquefying starch in a slurry, the slurry having been produced from a substrate comprising plant material in either a dry or wet milling process, said method comprising: a primary and/or secondary liquefaction step comprising adding to the slurry in the primary or secondary liquefaction step, in any order, a combination of at least one phytic acid hydrolyzing enzyme and at least one α-amylase variant, either simultaneously or separately to produce liquefied starch, wherein the α-amylase variant comprises an amino acid sequence having at least 90% sequence identity to the parental α-amylase polypeptide of SEQ ID NO: 2, and having a substitution that introduces the amino acid residue E188P, wherein the α-amylase variant exhibits increased viscosity reduction in a starch liquefaction assay compared to the parental polypeptide.
 33. The method of claim 32, wherein the starch is granular starch.
 34. The method of claim 32, further comprising saccharifying the liquefied starch to obtain fermentable sugars.
 35. The method of claim 34, further comprising fermenting the fermentable sugars under suitable fermentation conditions to obtain end-products such as alcohol.
 36. The method of claim 35, wherein the alcohol end-product is ethanol.
 36. The method of claim 32, wherein the phytic acid hydrolyzing enzyme is phytase.
 37. The method of claim 32, wherein the primary or secondary liquefaction step does not include pH adjustment of the slurry.
 38. The method of claim 37, wherein the pH of the primary or secondary liquefaction is in the range of pH 4.5 to 5.4.
 39. The method of claim 37, wherein the pH of the primary or secondary liquefaction is in the range of 4.8 to 5.8.
 40. A method of producing alcohol from starch slurry comprising: a primary and/or secondary liquefaction step comprising adding to the starch slurry in the primary or secondary liquefaction step, in any order, a combination of a phytic acid hydrolyzing enzyme and an α-amylase variant, either simultaneously or separately to produce liquefied starch, wherein the α-amylase variant comprises an amino acid sequence having at least 90% sequence identity to the parental α-amylase polypeptide of SEQ ID NO: 2, and having a substitution that introduces the amino acid residue E188P, wherein the α-amylase variant exhibits increased viscosity reduction in a starch liquefaction assay compared to the parental polypeptide, a saccharification step comprising saccharifying the liquefied starch to obtain fermentable sugars to produce fermentable sugars, fermenting the fermentable sugars under suitable fermentation conditions using a fermenting microorganism to produce alcohol.
 41. The method of claim 40 wherein the phytic acid hydrolyzing enzyme is phytase.
 42. The method of claim 40 wherein the alcohol is ethanol.
 43. The method of claim 40, wherein the primary or secondary liquefaction step does not include pH adjustment of the slurry.
 44. The method of claim 43, wherein the pH of the primary or secondary liquefaction is in the range of pH 4.5 to 5.4.
 45. The method of claim 43, wherein the pH of the primary or secondary liquefaction is in the range of 4.8 to 5.8. 